Stable cell lines and methods for evaluating gastrointestinal absorption of chemicals

ABSTRACT

Nucleic acids and vectors for interfering with the expression of membrane efflux transport proteins in cells that express such proteins are provided. Also provided are cells and cell lines comprising such nucleic acids and vectors. Methods for screening chemicals and biomolecules for gastrointestinal absorption in animals, and kits for practicing such methods are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/938,065, filed Nov. 9, 2007, now allowed, which claims the benefit ofU.S. Provisional Application No. 60/892,665, filed Mar. 2, 2007, and ofU.S. Provisional Application No. 60/857,938, filed Nov. 10, 2006, all ofwhich are incorporated by reference herein.

FIELD

The invention relates generally to the field of pharmacology. Morespecifically, the invention features stable cell lines, kits, andmethods for predicting the absorption of chemicals such as drugs,nutritional supplements, and environmental chemicals upon administrationto animals or humans.

BACKGROUND

Various publications, including patents, published applications,technical articles and scholarly articles are cited throughout thespecification. Each of these cited publications is incorporated byreference herein, in its entirety and for all purposes.

Drug absorption is the sum total of the effects of various mechanisms bywhich drugs pass from the point of entry into the bloodstream. The rateand efficiency of drug absorption affects the rate and extent to which adrug reaches its intended site of action. Gastrointestinal absorption oforally administered drugs is, in part, a function of the permeability ofmucosa in the gastrointestinal tract, particularly in the intestines,and also, in part, a function of the transit rate through the variousorgans of the gastrointestinal tract as the transit rate establishes thelength of time the drug is localized to an absorption site.

Intestinal absorption of drugs can occur via different routes. Manyorally administered drugs are absorbed by passive transcellulardiffusion through the cell membrane of enterocytes (Van Asperen J et al.(1998) Pharm. Res. 37:429-35) or by passive paracellular diffusionthrough the tight junctions in the intestinal epithelium (Watson C J etal. (2001) Am. J. Physiol. Cell Physiol. 281:C388-C397). Epithelial drugabsorption is also mediated by membrane transport proteins (Lee V H(2001) J. Natl. Cancer Inst. Monogr. 29:41-4). Such transport proteinscan also serve as an impediment to drug absorption.

Various efflux transporters have been described, including members ofthe multidrug resistance protein (MRP) family (Borst P et al. (2000) J.Natl. Cancer Inst. 92:1295-1302), P-glycoprotein (P-gp) (Germann U A(1996) Eur. J. Cancer 32A:927-44), and breast cancer resistance protein(BCRP) (Staud F et al. (2005) Int. J. Biochem. Cell Biol. 374:720-5;Doyle L A et al. (1998) Proc. Natl. Acad. Sci. USA. 9526:15665-70),among others. Such efflux transport proteins are believed to beprimarily responsible for low or variable absorption of orallyadministered drugs (Stephens R H et al. (2001) J. Pharmacol. Exp. Ther.296:584-91).

Drug absorption and the factors that facilitate or impede it are thusimportant considerations in drug design and the evaluation of leadcompounds as potential therapeutic agents. Several models are availablefor assessing absorption in the intestine. These models include theparallel artificial membrane permeability assay (PAMPA), in situintestinal recirculating and single-pass perfusion, Ussing chambers, andcell lines, including Madin-Darby canine kidney cells (MDCK), and Caco-2cells (Balimane P V et al. (2006) AAPS J. 8:E1-13).

Caco-2 cells, which were derived from a human colon adenocarcinoma, area widely used model for intestinal absorption studies. When grown andallowed to differentiate, Caco-2 cells are morphologically similar toenterocytes and express many of the enzymes present in the smallintestinal brush border, and thus closely resemble the environment andfunctions of the small intestine. Caco-2 cells provide an additionaladvantage for intestinal absorption studies in that they express atleast three drug efflux transporter proteins, including P-gp (Hunter Jet al. (1993) J. Biol. Chem. 268:14991-7), MRP proteins (Hirohashi T etal. (2000) J. Pharmacol. Exp. Ther. 292:265-70; Gutmann H et al. (1999)Pharm. Res. (NY) 16:402-7), and BCRP (Xia C Q et al. (2005) Drug Metab.Dispos. 33:637-43).

For drug absorption studies, it is desirable to evaluate thecontributions of drug efflux transport proteins to impaired absorption.This can be accomplished by inhibiting the expression or activity of thetransporters, particularly P-gp. In general, chemicals such ascyclosporine A are used to block the activity of P-gp. Chemicalinhibition of transporters presents a disadvantage insofar as suchchemicals also inhibit other cellular proteins and functions, and thuscan skew the results of absorption experiments. Recently, the expressionof P-gp in Caco-2 cells was shown to be reduced using RNAi technology(Watanabe T et al. (2005) Pharm. Res. 22:1287-93), and the expression ofmultidrug resistant gene 1 (MDR1) in Caco-2 cells was shown to bereduced using RNAi technology (Celius T et al. (2004) Biochem. Biophys.Res. Comm. 324:365-71). Inhibition of P-gp expression by RNAi enhancedthe intracellular accumulation of and restored the sensitivity tocompounds transported by P-gp (Wu H et al. (2003) Cancer Res.63:1515-19). While genetically down-regulating P-gp in Caco-2 cellsrepresents an improvement over the use of chemical inhibitors, studiesof drug absorption in this model are disadvantaged in that the knockdownof P-gp expression alone does not account for contributions of extanttransporters such as MRP and BCRP to drug efflux and impairedabsorption. In addition, shRNA synthesized in vitro and directlytransfected into cells reduces gene expression only transiently, andexpression is restored a few days after transfection. Moreover, invitro-synthesized shRNA is also often limited to cells that are easilytransfected, and very little is known about the stability of inhibitionof gene expression after several cell passages.

To accurately evaluate and predict the intestinal absorption of leadcompounds, it is desired that the relative contributions of any and allefflux transport proteins be accounted for and controlled. Similarly, itis desirable to produce and utilize stable cell lines to geneticallycontrol the expression and/or function of such transport proteins on amore permanent basis. The present invention addresses these long-feltneeds.

SUMMARY

The invention features isolated nucleic acid molecules for inhibitingexpression of at least one membrane efflux transport protein, thenucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, 2, 3, 4, 5, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 or allelicvariants thereof. The nucleic acid molecules can be single, double, ortriple stranded. In some preferred aspects, the nucleic acid moleculesare RNA.

Vectors comprising nucleic acid sequences encoding a nucleic acidmolecule for inhibiting expression of a membrane efflux transportprotein, wherein said nucleic acid molecule comprises SEQ ID NO: 1, 2,3, 4, 5, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26, or allelic variantsthereof are also provided. In the vectors, the nucleic acid molecule canbe operably linked to one or more regulatory elements such as aninducible or constitutive promoter. The vectors can be viral vectors,and in some preferred embodiments are lentivirus vectors.

Host cells transformed with such vectors are also provided by thisinvention. It is preferred that such host cells chosen fortransformation with the vectors express at least one membrane effluxtransport protein such that transformation will result in inhibition ofthe expression of the protein. The membrane efflux transport protein canbe any such protein. Examples of membrane efflux transport proteinsinclude P-glycoprotein, Multidrug Resistance-Associated Protein 2, andBreast Cancer Resistance Protein. Host cells can be epithelial cells,and are preferably intestinal epithelial cells, and are more preferablyhuman intestinal epithelial cells. Examples of suitable cells includeCaco-2 cells, C2BBe1 cells, HT-29 cells, and T-84 cells. Host cellcultures are also provided.

Also featured are methods for screening compounds for gastrointestinalabsorption in an animal such as a human comprising stably inhibiting theexpression of at least one membrane efflux transport protein in a cell,contacting the cell with a test compound, measuring transcellulartransport of the test compound, and comparing the transcellulartransport measurements with reference values for transcellular transportof compounds with no gastrointestinal absorption, low gastrointestinalabsorption, moderate gastrointestinal absorption, or highgastrointestinal absorption. The measurements relative to the referencevalues are indicative of the gastrointestinal absorption of the compoundin the body of the animal Examples of membrane efflux transport proteinsinclude P-glycoprotein, Multidrug Resistance-Associated Protein 2, andBreast Cancer Resistance Protein.

In such methods, the inhibiting can comprise stably transforming thecell with a nucleic acid molecule that interferes with the expression ofthe at least one membrane efflux transport protein. Examples of suitablecells include Caco-2 cells, C2BBe1 cells, HT-29 cells, and T-84 cells.Suitable nucleic acid molecules include those having SEQ ID NO: 1, 2, 3,4, 5, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26, and conservativelymodified variants or allelic variants thereof. One non-limiting exampleof stable transformation involves the use of viral, and preferablylentiviral vectors that comprise the nucleic acid molecule. In someaspects, a single nucleic acid molecule can inhibit the expression oftwo or more membrane efflux transport proteins.

Also featured are methods for screening compounds for gastrointestinalabsorption in an animal such as a human comprising stably inhibiting theexpression of a first membrane efflux transport protein in a cell,stably inhibiting the expression of a second membrane efflux transportprotein in a second cell, contacting the first and second cells with atest compound, measuring transcellular transport of the test compound inthe first and second cells, and comparing the transcellular transportmeasurements with reference values for transcellular transport ofcompounds with no gastrointestinal absorption, low gastrointestinalabsorption, moderate gastrointestinal absorption, or highgastrointestinal absorption. In some embodiments, the methods furthercomprise stably inhibiting the expression of a third membrane effluxtransport protein in a third cell, contacting the first, second, andthird cells with a test compound, measuring transcellular transport ofthe test compound in the first, second, and third cells, and comparingthe transcellular transport measurements with reference values fortranscellular transport of compounds with no gastrointestinalabsorption, low gastrointestinal absorption, moderate gastrointestinalabsorption, or high gastrointestinal absorption. For each comparison,the measurements indicate the relative contribution of the first,second, and third membrane efflux transport protein to transcellulartransport of the test compound, and the measurements relative to thereference values are predictive of the gastrointestinal absorption ofthe compound in the body of the animal. Examples of membrane effluxtransport proteins include P-glycoprotein, MultidrugResistance-Associated Protein 2, and Breast Cancer Resistance Protein.

In such methods, the inhibition of the first, second, and/or thirdmembrane efflux transport protein can comprise transforming the cellwith a nucleic acid molecule that interferes with the expression of theat least one membrane efflux transport protein. Examples of suitablecells include Caco-2 cells, C2BBe1 cells, HT-29 cells, and T-84 cells.Suitable nucleic acid molecules include those having SEQ ID NO: 1, 2, 3,4, 5, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26, and conservativelymodified variants or allelic variants thereof. One non-limiting exampleof stable inhibition involves the use of viral, and preferablylentiviral vectors that comprise the nucleic acid molecule. In someaspects, a single nucleic acid molecule can inhibit the expression oftwo or more membrane efflux transport proteins.

The invention also provides methods for inhibiting the expression of amembrane efflux transport protein. The methods comprise stablytransforming a cell with a vector comprising a nucleic acid sequenceencoding a nucleic acid molecule that interferes with the expression ofa membrane efflux transport protein, and expressing said nucleic acidmolecule in the cell, wherein expression of the nucleic acid moleculeinhibits the expression of the membrane efflux transport protein.Lentivirus vectors are preferred. The membrane efflux transport proteincan be P-glycoprotein, Multidrug Resistance-Associated Protein 2, orBreast Cancer Resistance Protein. The nucleic acid sequence can compriseSEQ ID NO: 1, 2, 3, 4, 5, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26, orconservatively modified variants or allelic variants thereof. Onenon-limiting example of stable transformation involves the use of viral,and preferably lentiviral vectors that comprise the nucleic acidmolecule. In some aspects, a single nucleic acid molecule can inhibitthe expression of two or more membrane efflux transport proteins.

Kits for screening compounds for gastrointestinal absorption in animalsare provided by the invention. In some embodiments, such kits cancomprise, in one or more containers, a cell stably transformed with atleast one nucleic acid molecule for inhibiting expression of at leastone membrane efflux transport protein and instructions for using the kitin a method for screening compounds for gastrointestinal absorption inanimals. The kits can further comprise additional cells stablytransformed with a nucleic acid molecule that interferes with theexpression of additional membrane efflux transport protein. Examples ofmembrane efflux transport proteins include P-glycoprotein, MultidrugResistance-Associated Protein 2, and Breast Cancer Resistance Protein.Examples of suitable cells include Caco-2 cells, C2BBe1 cells, HT-29cells, and T-84 cells. Suitable nucleic acid molecules include thosehaving SEQ ID NO: 1, 2, 3, 4, 5, 17, 18, 19, 20, 21, 22, 23, 24, 25, or26, and conservatively modified variants or allelic variants thereof.

The invention also features methods for identifying compounds thatinhibit the efflux activity of a membrane efflux transport protein. Suchmethods, in some embodiments, comprise stably inhibiting the expressionof a membrane efflux transport protein in a first cell, contacting thefirst cell with a substrate of the membrane efflux transport protein,contacting a second cell expressing the membrane efflux transportprotein with a test compound and a substrate of the membrane effluxtransport protein, determining the efflux activity of the membraneefflux transport protein in the first cell, and in the second cell inthe presence and absence of the test compound, and comparing thedetermined efflux activities, wherein a decrease in the efflux activityin the presence of the test compound relative to the efflux activity inthe absence of the test compound, and at least partial identity of theefflux activity in the presence of the test compound with the effluxactivity in the first cell indicates that the test compound specificallyinhibits the membrane efflux transport protein.

In such methods, the stable inhibition of the membrane efflux transportprotein in the first cell can comprise transforming the cell with anucleic acid molecule that interferes with the expression of the atleast one membrane efflux transport protein. Examples of membrane effluxtransport proteins include P-glycoprotein, MultidrugResistance-Associated Protein 2, and Breast Cancer Resistance Protein.Suitable nucleic acid molecules include those having SEQ ID NO: 1, 2, 3,4, 5, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26, and conservativelymodified variants or allelic variants thereof. Examples of cells thatcan serve as the first and/or second cell include Caco-2 cells, C2BBe1cells, HT-29 cells, and T-84 cells. One non-limiting example of stableinhibition involves the use of viral, and preferably lentiviral vectorsthat comprise the nucleic acid molecule. In some aspects, a singlenucleic acid molecule can inhibit the expression of two or more membraneefflux transport proteins.

Any known substrate of the membrane efflux transport protein of interestcan be used in the methods. Examples of such substrates include digoxinfor P-glycoprotein, vinblastine or dinitrophenyl-5-glutathione forMultidrug Resistance-Associated Protein 2, and mitoxantrone orestrone-3-sulfate for Breast Cancer Resistance Protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows P-gp function was significantly decreased in cellstransduced with lentiviruses containing SEQ ID NOs: 1, 2, 3, 4, or 5(shRNA/P-gp 83, 84, 85, 86, or 87, respectively) as described herein, asdetermined by intracellular calcein fluorescence. shRNA/P-gp clone cells(KD) were treated in parallel with Cyclosporin A (CsA) (KD+CsA), anestablished P-gp inhibitor. CsA was used as a positive control(C2BBe1+CsA), and unknockdown, non-transduced C2BBe1 cells (Un-KD) wereused as a negative control to show baseline calcein retention.

FIG. 2 shows gel electrophoretic analysis of RT-PCR amplified human P-gpmRNA from shRNA/P-gp clone cells and control cells. Non-transducedC2BBe1 (un-knockdown C2BBE1 cells) and MDR1/MDCK cells, shown in laneslabeled 1 and 2, respectively, prominently display a 208 base pair (bp)band corresponding to P-gp (top panel). In contrast, cells transducedwith lentiviruses containing SEQ ID NOs: 1, 2, 3, 4, or 5 (shRNA/P-gp83, 84, 85, 86, or 87, respectively) show significantly reducedexpression of P-gp mRNA. β-actin is shown as a positive control (bottompanel). M1=100 by DNA marker.

FIG. 3 shows the efflux ratio of digoxin in shRNA/P-gp clone cells. Theefflux ratio (Papp B-A:A-B) is significantly reduced in cells transducedwith lentiviruses containing SEQ ID NOs: 1, 2, 3, 4, or 5 (shRNA/P-gp83, 84, 85, 86, or 87, respectively) relative to non-transformed C2BBe1cells (Un-KD).

FIG. 4 shows a Western blot analysis of P-gp protein expression inC2BBe1 cells transduced with lentiviruses containing SEQ ID NOs: 1, 2,3, 4, or 5 (shRNA/P-gp 83, 84, 85, 86, or 87, respectively). Transducedcells demonstrated significantly reduced P-gp protein expressionrelative to non-transduced C2BBe1 cells (C) run in parallel. P-gpexpressed in insect cell microsomes was used as a positive control(P-gp). MDCK cell extract, which does not express human P-gp, was usedas a negative control (M). β-actin was blotted as a standard fornormalization of the amount of proteins transferred to the blottingmembrane.

FIG. 5 shows the calculated percent inhibition of P-gp proteinexpression in C2BBe1 cells transduced with lentiviruses containing SEQID NOs: 1, 2, 3, 4, or 5 (shRNA/P-gp 83, 84, 85, 86, or 87,respectively), relative to non-transduced C2BBe1 cells, as determined byWestern blot.

FIG. 6 shows MRP2 mRNA expression in C2BBe1 shRNA/MRP2 clone cells #3,4, 5, 6, and 7 (transduced with SEQ ID NOs 17, 18, 19, 20, and 21,respectively). Vector control (VC) cells were transduced with a controlshRNA vector described in Example 7. Total cellular RNA from cells withpassages ranging from 2 to 5 was isolated after 5 to 7 days of growth,amplified with MRP2-specific primers (SEQ ID NOs: 11 and 12), andresolved by agarose gel electrophoresis. β-actin was included to accountfor varying efficiencies of total RNA extraction from the cell extracts.

FIG. 7 shows Western blot analysis of MRP2 protein levels in C2BBe1shRNA/MRP2 clone cells #3, 4, 5, 6, and 7 (transduced with SEQ ID NOs:17, 18, 19, 20, and 21, respectively). Vector control (VC) cells weretransduced with a control shRNA vector described in Example 7. Theβ-actin was included to account for variations in the amount of totalprotein applied to the electrophoresis gel.

FIG. 8 shows BCRP mRNA expression in C2BBe1 cells transduced withlentiviruses containing SEQ ID NOs: 22, 23, 24, 25, or 26 (shRNA/BCRP798, 799, 800, 801, or 802, respectively). Transduced C2BBe1 cells werecultured and grown, and total cellular RNA was extracted. Extracted RNAwas amplified by RT-PCR with BCRP-specific primers (SEQ ID NOs: 13 and14), and the products were resolved by agarose gel electrophoresis. Allfive shRNA/BCRP inserts significantly reduced BCRP mRNA expressionrelative to C2BBe1 cells transduced with a control lentivirus containingnon-interfering shRNA (VC). β-actin mRNA production was also assessed toaccount for varying efficiencies of total RNA extraction from the cellextracts.

FIG. 9 shows relative levels of BCRP mRNA expression in C2BBe1 cellstransduced with lentiviruses containing SEQ ID NOs: 22, 23, 24, 25, or26 (shRNA/BCRP 798, 799, 800, 801, or 802, respectively). TransducedC2BBe1 cells were cultured and grown, and total cellular RNA wasextracted. RT-PCR was carried out, and the products were resolved byagarose gel electrophoresis (FIG. 8). BCRP mRNA expression levels werecompared with the level of BCRP mRNA expression in C2BBe1 cellstransduced with a control lentivirus containing non-interfering shRNA(VC) to determine the degree of inhibition of BCRP mRNA expressioncaused by interfering shRNAs. Results are illustrated as percentinhibition of BCRP mRNA expression.

FIG. 10 shows expression of BCRP protein in C2BBe1 cells transduced withlentiviruses containing SEQ ID NOs: 22, 23, 24, 25, or 26 (shRNA/BCRP798, 799, 800, 801, or 802, respectively). Western blot analysisindicated a substantial decrease in the amount of BCRP protein presentin shRNA/BCRP clone cells #798, 799, 800, 801, and 802 (cell passage 15to 16) as compared to C2BBe1 cells transduced with a control lentiviruscontaining non-interfering shRNA (VC). The β-actin was included toaccount for variations in the amount of total protein applied to theelectrophoresis gel.

FIG. 11 shows percent inhibition of BCRP protein expression inshRNA/BCRP clone cells #798, 799, 800, 801, and 802 (as shown in FIG.10). The ratio of optical densities for P-gp and beta-actin bands shownin FIG. 10 was used to determine percent inhibition of BCRP proteinexpression in shRNA/BCRP clone cells relative to expression levels inC2BBe1 cells transduced with a control lentivirus containingnon-interfering shRNA.

FIG. 12 shows expression of BCRP mRNA in shRNA/BCRP clone cell line #801from cell passages 5 to 20 as measured by RT-PCR. Expression of BCRPmRNA decreased from passage 5 to passage 20. Amplified mRNAs wereseparated and visualized as described in Example 1. β-actin was includedto account for varying efficiencies of total RNA extraction from thecell extracts.

FIG. 13 shows expression of P-gp mRNA and MRP2 mRNA in shRNA/BCRP clone801 at passages 10 and 20. Expression of P-gp mRNA in shRNA/BCRP clone801 was increased compared to vector control cells (VC), non-transducedC2BBe1 (Wt) at both passages 10 and 20. In contrast to P-gp, MRP2 mRNAshowed significantly decreased expression in clone #801 cells comparedto the other cell lines tested only at passage 20. β-actin was includedto account for varying efficiencies of total RNA extraction from thecell extracts.

FIG. 14 shows the corresponding Western blot to FIG. 13 for theexpression of BCRP, P-gp and MRP2 proteins in shRNA/BCRP clone cell line#801 for cell passages 10 and 20. The MRP2 band is present in shRNA/BCRPclone 801 passage 10, but not passage 20. The β-actin was included toaccount for variations in the amount of total protein applied to theelectrophoresis gel.

DETAILED DESCRIPTION

Various terms relating to the methods and other aspects of the presentinvention are used throughout the specification and claims. Such termsare to be given their ordinary meaning in the art to which the inventionpertains, unless otherwise indicated. Other specifically defined termsare to be construed in a manner consistent with the definition providedherein. Although any methods and materials similar or equivalent tothose described herein can be used in the practice for testing of thepresent invention, the preferred materials and methods are describedherein.

The following abbreviations are used throughout the specification. Papp:Permeability coefficient; PappA-B: Permeability coefficient in theapical (A) to basolateral (B) direction; PappB-A: Permeabilitycoefficient in the basolateral (B) to apical (A) direction; ER: Effluxratio, PappB-A/PappA-B ratio; P-gp: P-glycoprotein; MDR1: Multi-drugresistance protein 1; BCRP: Breast cancer resistance protein; MRP:Multi-drug resistance-associated protein; MRP2: Multi-drugresistance-associated protein 2; DMEM, Dulbecco's Modified Eagle Medium;FBS, fetal bovine serum; FTC: Fumitremorgin C; CsA: Cyclosporin A;MK571: MRP Inhibitor; TEER, transepithelial electrical resistance; KD:Knocked-down; WT: Wild-type, unmodified parental cell line; C2BBe1WT:Unmodified C2BBe1 cells; C2BBe1 Pgp-KD: C2BBe1 cells in which theexpression of Pgp has been suppressed; C2BBe1 BCRP-KD: C2BBe1 cells inwhich the expression of BCRP has been suppressed; C2BBe1 MRP2-KD: C2BBe1cells in which the expression of MRP2 has been suppressed; MDCK:Madin-Darby canine kidney; MDR1-MDCK: MDCK cell line transfected withthe human MDR1 gene, which overexpresses; BCRP-MDCK: MDCK cell linetransfected with the human BCRP gene, which overexpresses; MRP2-MDCK:MDCK cell line transfected with the human MRP2 gene, whichoverexpresses; nt, nucleotide.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a cell”includes a combination of two or more cells, and the like.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

“Isolated” means altered “by the hand of man” from the natural state. Ifa molecule or composition occurs in nature, it has been “isolated” if ithas been changed or removed from its original environment, or both. Forexample, a polynucleotide or a polypeptide naturally present in a livingplant or animal is not “isolated,” but the same polynucleotide orpolypeptide separated from the coexisting materials of its natural stateis “isolated” as the term is employed herein.

“Polynucleotide,” synonymously referred to as “nucleic acid molecule,”refers to any polyribonucleotide or polydeoxyribonucleotide, which maybe unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides”include, without limitation single- and double-stranded DNA, DNA that isa mixture of single- and double-stranded regions, single- anddouble-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions. In addition, “polynucleotide”refers to triple-stranded regions comprising RNA or DNA or both RNA andDNA. The term polynucleotide also includes DNAs or RNAs containing oneor more modified bases and DNAs or RNAs with backbones modified forstability or for other reasons. “Modified” bases include, for example,tritylated bases and unusual bases such as inosine. A variety ofmodifications can be made to DNA and RNA; thus, “polynucleotide”embraces chemically, enzymatically or metabolically modified forms ofpolynucleotides as typically found in nature, as well as the chemicalforms of DNA and RNA characteristic of viruses and cells.“Polynucleotide” also embraces relatively short nucleic acid chains,often referred to as oligonucleotides.

A “vector” is a replicon, such as plasmid, phage, cosmid, or virus towhich another nucleic acid segment may be operably inserted so as tobring about the replication or expression of the segment.

The term “express,” “expressed,” or “expression” of a nucleic acidmolecule refers to the biosynthesis of a gene product. The termencompasses the transcription of a gene into RNA. For example, but notby way of limitation, a regulatory gene such as an antisense nucleicacid or interfering nucleic acid can be expressed by transcription asantisense RNA or RNAi or shRNA. The term also encompasses translation ofRNA into one or more polypeptides, and encompasses all naturallyoccurring post-transcriptional and post-translational modifications.

The term “operably linked” or “operably inserted” means that theregulatory sequences necessary for expression of the coding sequence areplaced in a nucleic acid molecule in the appropriate positions relativeto the coding sequence so as to enable expression of the codingsequence. By way of example, a promoter is operably linked with a codingsequence when the promoter is capable of controlling the transcriptionor expression of that coding sequence. Coding sequences can be operablylinked to promoters or regulatory sequences in a sense or antisenseorientation. The term “operably linked” is sometimes applied to thearrangement of other transcription control elements (e.g., enhancers) inan expression vector.

A “heterologous” region of a nucleic acid construct is an identifiablesegment (or segments) of the nucleic acid molecule within a largermolecule that is not found in association with the larger molecule innature. Thus, when the heterologous region encodes a mammalian gene, thegene will usually be flanked by DNA that does not flank the mammaliangenomic DNA in the genome of the source organism.

A cell has been “transformed” or “transduced” by exogenous orheterologous nucleic acids such as DNA when such DNA has been introducedinside the cell. The transforming DNA may or may not be integrated(covalently linked) into the genome of the cell. In prokaryotes, yeast,and mammalian cells for example, the transforming DNA may be maintainedon an episomal element such as a plasmid. With respect to eukaryoticcells, a stably transformed cell, or “stable cell” is one in which thetransforming DNA has become integrated into a chromosome so that it isinherited by daughter cells through chromosome replication. Thisstability is demonstrated by the ability of the eukaryotic cell toestablish cell lines or clones comprised of a population of daughtercells containing the transforming DNA. A “clone” is a population ofcells derived from a single cell or common ancestor by mitosis. A “cellline” is a clone of a primary cell that is capable of stable growth invitro for many generations.

As used herein, “test compound” refers to any purified molecule,substantially purified molecule, molecules that are one or morecomponents of a mixture of compounds, or a mixture of a compound withany other material that can be analyzed using the methods of the presentinvention. Test compounds can be organic or inorganic chemicals, orbiomolecules, and all fragments, analogs, homologs, conjugates, andderivatives thereof “Biomolecules” include proteins, polypeptides,nucleic acids, lipids, monosaccharides, polysaccharides, and allfragments, analogs, homologs, conjugates, and derivatives thereof. Testcompounds can be of natural or synthetic origin, and can be isolated orpurified from their naturally occurring sources, or can be synthesizedde novo. Test compounds can be defined in terms of structure orcomposition, or can be undefined. The compound can be an isolatedproduct of unknown structure, a mixture of several known products, or anundefined composition comprising one or more compounds. Examples ofundefined compositions include cell and tissue extracts, growth mediumin which prokaryotic, eukaryotic, and archaebacterial cells have beencultured, fermentation broths, protein expression libraries, and thelike.

“Membrane efflux transport protein” refers to any protein transporterslocalized to a cell membrane. Such transport proteins can have as one oftheir biological functions the ability to mediate the removal ofcompounds from the cell interior, herein referred to as “effluxactivity.” Efflux activity can result in broad substrate specificityresistance to multiple structure-unrelated therapeutic agents, i.e.,multidrug resistance (MDR). The ability of membrane efflux transportprotein to confer clinical MDR has generated considerable interest inidentifying the substrates and/or inhibitors of such protein and soreversing innate or acquired drug resistance (N. Mizuno, et al. (2003)Pharmacological Rev. 55:425-61).

As used herein, the term “modulate” means any change, enhancement orinhibition in the amount, quality, or activity of a particularbiomolecule or pathway. “Inhibit” or “inhibition” or “interfere” meansto reduce, decrease, block, prevent, delay, inactivate, desensitize,stop, or downregulate the biological activity or expression of amolecule, protein or pathway of interest. In some preferred embodimentsof the invention, the level of the expression or biological activity ofa protein or pathway of interest, for example, efflux activity orexpression of membrane efflux proteins, refers to a decrease (inhibitionor downregulation) or increase (upregulation) of greater than from about50% to about 99%, and more specifically, about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69% 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more. The inhibition may be direct, i.e., operate onthe molecule or pathway of interest itself, or indirect, i.e., operateon a molecule or pathway that affects the molecule or pathway ofinterest.

“Knockdown” refers to a cell or organism having reduced expression ofone or more genes. As will be appreciated by those skilled in the art, aknockdown will exhibit at least about a 20% reduction in expression,preferably will exhibit at least about a 50% reduction in expression,and more preferably will exhibit at least about a 75% reduction inexpression, although higher reductions are possible, including at leastabout a 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more reduction in expression.

With respect to nucleic acids, the term “percent identity” refers to thepercentage of sequence identity found in a comparison of two or morenucleic acid sequences.

“Gastrointestinal absorption” refers to the uptake of chemicals,including biomolecules and test compounds, into or across tissues thatcomprise the gastrointestinal tract. For example, absorption includes,but is not limited to, uptake of compounds from the apical side of acell and the release of compounds from the basolateral side of a cell.The gastrointestinal tract comprises the stomach, small intestine, andlarge intestine. “No gastrointestinal absorption” means 0% of thecompound is absorbed. “Low gastrointestinal absorption” means that morethan 0%, but less than 25% of a compound is absorbed. “Moderategastrointestinal absorption” means that greater than or equal to 25% butless than 85% of a compound is absorbed. “High gastrointestinalabsorption” means that greater than or equal to 85% of a compound isabsorbed.

As used herein, “measure” or “determine” refers to any qualitative orquantitative determinations.

“Transcellular transport” refers to the movement of a compound across alayer of epithelial cells whereby the compound is moved through thecells and not the spaces between cells such as tight junctions. By wayof contrast, “paracellular transport” refers to the movement of acompound across a layer of epithelial cell whereby the compound is movedthrough the tight junctions between cells.

The following sections set forth the general procedures involved inpracticing the present invention. To the extent that specific materialsare mentioned, it is merely for the purpose of illustration, and is notintended to limit the invention. Unless otherwise specified, generalbiochemical and molecular biological procedures, such as those set forthin Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &Sons, New York, 1998; Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL, 2D ED., Cold Spring Harbor Laboratory Press, Plainview, N.Y.,1989; Kaufman et al., Eds., HANDBOOK OF MOLECULAR AND CELLULAR METHODSIN BIOLOGY AND MEDICINE, CRC Press, Boca Raton, 1995. PROTEINEXPRESSION: A PRACTICAL APPROACH (S J Higgins and B. D Hames, Eds.)Oxford University Press, Oxford, UK 1999; Charles Hardin et al. CLONING,GENE EXPRESSION, AND PROTEIN PURIFICATION: EXPERIMENTAL PROCEDURES ANDPROCESS RATIONALE Oxford University Press, Inc. New York, N.Y. 2001; andMEMBRANE PROTEIN PROTOCOLS: EXPRESSION, PURIFICATION, ANDCHARACTERIZATION (METHODS IN MOLECULAR BIOLOGY (Clifton, N.J.), V. 228.)Barry Steven Selinsky, Humana Press, Inc. Totowa, N.J. 2003 are used.

It is to be understood that this invention is not limited to particularmethods, reagents, compounds, compositions or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting.

Efflux transport proteins, particularly those expressed by intestinalcells, can limit the absorption of substrate drugs, and can mediatedrug-drug interactions that alter, frequently reducing, drugavailability or efficacy. Thus, understanding the role of the variousefflux transporters in drug absorption is important in the developmentof lead compounds. To date, the study of efflux transporters has largelyrelied on the use of chemical inhibitors. However, the use of chemicalinhibitors is problematic as the inhibitors are not specific and canaffect other cellular processes thereby blurring the overall picture ofdrug absorption. It has been discovered in accordance with the presentinvention that the expression of the various efflux transport proteinscan be stably inhibited at the genetic level using virally-transformedcells. It has further been discovered that targeted knockdown of thevarious transporters permits the identification of the particulartransporters involved in drug transport with a high degree of certainty.The knockdown approach described herein provides stable,sequence-specific silencing of membrane efflux transport proteinsinduced by endogenous expression of shRNA by lentiviral vectors. Theinvention is advantageous over previous knockdown attempts as thetransformation with lentiviral vectors provides permanent, stableinhibition of gene expression, and provides the additional advantage ofcircumventing non-specific inhibition of other cellular functions thatwould be expected from chemical inhibition.

Accordingly, in one aspect, the invention features methods for screeningtest compounds for gastrointestinal absorption in animals. The methodscomprise modulating, and preferably inhibiting, the expression of atleast one membrane efflux transport protein in a cell, contacting thecell with a test compound, measuring transcellular transport of the testcompound, and comparing the transcellular transport measurements withreference values for transcellular transport of compounds with nogastrointestinal absorption, low gastrointestinal absorption, moderategastrointestinal absorption, or high gastrointestinal absorption. Thevalues obtained by experiments measuring the transcellular transport ofthe compound indicate the degree to which the test compound is likely tobe absorbed upon administration to the animal. The methods are thususeful as in vitro models, among other things.

Membrane efflux transport proteins, synonymously referred to herein asdrug transporters or drug transport proteins, generally are comprised ofone or more subunits that span the plasma membrane of mammalian cells,including mammalian epithelial cells. Mammalian epithelial cells areoften polarized cells, meaning that their membrane composition differsbetween the apical or outward facing portion of the cell, and thebasolateral or inward portion of the cell. By apical or outward, it ismeant that such portions of the cell face a body compartment connectedwith the outside environment, such as the lumen of the intestine or thelining of the urinary tract or the bile duct. By basolateral or inward,it is meant that such portions of the cell face the interior of thebody, typically the body's blood supply. Epithelial cells play a role inabsorption and elimination of nutrients, drugs and environmental toxinsas well as metabolites derived from compounds in each of thesecategories. Epithelial cells can grow together in sheets in whichneighboring cells are linked together by tight junctions, which areintercellular connections that limit the diffusion of ions and largermolecules between cells, called paracellular transport.

Liver hepatocytes and brain capillary endothelial cells share certaincommon features with epithelial cells, including the presence of tightjunctions and asymmetric expression of membrane transport proteins. Manyepithelial cells maintain this asymmetrical orientation even whencultured outside the body. This can require special in vitro cultureconditions, such as the use of collagen-coated plastic ware, or theaddition of one or more differentiation-inducing proteins or hormones tothe cell culture medium. Other specialized culture conditions are knownto and readily practiced by those of skill in the art.

Membrane efflux transporters can be distributed asymmetrically inepithelial cells. This asymmetrical distribution can lead to vectorialtransport of compounds that are substrates for such transporters.Vectorial transport means that the rate of transport of a compounddiffers significantly depending on whether the compound is applied tothe apical or basolateral surface of an epithelial cell layer.

Mammalian membrane transporters can be divided into two general familiesbased on their gene sequence relationships and modes of compoundtransport. The first class is referred to as ABC transporters. The term“ABC” refers to a common structural feature of this family oftransporters, the presence of an Adenosine triphosphate (A) binding (B)cassette (C) structural motif. These transport proteins typically movechemicals from inside of a cell or from within the phospholipid bilayerof the cell membrane to outside the cell against an unfavorableconcentration gradient by using the energy provided by the hydrolyticcleavage of adenosine triphosphate (ATP) to adenosine diphosphate (ADP)and phosphate ion (Pi). Without intending to be limited to anyparticular theory or mechanism of action, it is believed that, inaddition to their roles as drug transporters, members of the ABC familyplay a role in excretion of bile salts and phospholipids into the bileas well as regulating the phospholipid and cholesterol content ofcellular membranes.

The second major family of transporters present in mammals is referredto as SLC family where the abbreviation “SLC” refers to the majorcharacteristics of this family of “Solute Linked Carriers,” namely thattransport of drugs into or out of a cell is linked to transport of aphysiological solute, such as sodium ion, proton or metabolic product,in either the opposite direction, via solute exchange, or in the samedirection, via co-transport. Unlike the ABC family of transporters, theSLC family generally does not require energy derived from ATP hydrolysisto function. Instead, such transporters utilize the concentrationgradients of the co-transported and/or exchanged molecules or ions as anenergy source for transporting drugs against an unfavorableconcentration gradient. Also, unlike ABC family transporters, SLCfrequently mediate compound uptake into cells. In some cases, such as inkidney tubule epithelial cells, SLC and ABC transporters are present onopposite sides of the polarized epithelium and work in concert todeliver compounds from the blood into the urine against unfavorableconcentration gradients (Wright S. et al. (2004) Physiol. Rev84:987-1049).

Table 1, below, lists human members of the ABC transporter family andsome of their known substrates and inhibitors (Zhang L, et al. (2006)Molecular Pharmaceutics 3: 62-9). The list of substrates and inhibitorsis for illustration purposes, and is not intended to be exhaustive.

TABLE 1 ABC transport proteins and known substrates and inhibitorsTrans- porter Common Tissue Gene Aliases Distribution SubstratesInhibitors ABCB1 P-gp, Intestine, liver, Digoxin, Ritonavir, MDR1kidney, brain, fexofenadine, cyclosporine, placenta, indinavir,verapamil, adrenal, testes vincristine, erythromycin, colchicine,ketoconazole, topotecon, itraconazole, paclitaxel, quinidine, loperamideelacridar (GF120918), azithromycin, valspodar (PSC833), LY335979 ABCB11BSEP Liver Vinblastine ABCC1 MRP1 Intestine, liver, Adefovir, kidney,brain indinavir ABCC2 MRP2, Intestine, Indinavir, Cyclosporine CMOATkidney, liver, cisplatin brain ABCC3 MRP3, Intestine, liver, Etoposide,CMOAT2 kidney, methotrexate, placenta tenoposide ABCC6 MRP6 Liver,kidney Cisplatin, daunorubicin ABCG2 BCRP Intestine, liver,Duanorubicin, Elacridar breast, placenta doxorubicin, (GF120918),topotecan, fumitremorgin rosuvastatin, C, gefitinib sulfasalazine

The drug rifampin and the herbal supplement St. John's wort areestablished inducers of increased ABCB1 gene expression. The ABCtransporter family mediates transport of drugs from a wide variety oftherapeutic classes, including antineoplastic drugs, such as cisplatinand topotecan, anti-viral drugs, such as indinavir and adefovir and theanti-hyperlipidemic drug, rosuvastatin. There is considerable overlapamong substrates for different transporters. For example, indinavir is asubstrate for ABCB1, ABCC1, and ABCC2. Similarly, topotecan is asubstrate for ABCB1 and ABCG2. At least in the case of ABCB1, or P-gp asit is commonly referred to, several drugs from a wide range oftherapeutic classes can inhibit the transporter without being substratesof the transporter. Orthologs of these human transporters with varyingdegrees of gene sequence homology exist in other mammalian species, suchas rats, mice, dogs and non-human primates.

The inventive methods are applicable for analyzing test compounds fortheir gastrointestinal absorption in any animal, preferably areapplicable to mammals, including companion animals such as dogs, cats,rabbits, and are most preferably applicable to humans. As such, themethods can be carried out in any cell that is representative of ananimal or group of animals of interest. The cells can be freshlyisolated, established cell lines, or can be cell lines produced de novo.The cell preferably expresses at least one membrane efflux transportprotein, although in some embodiments, the cell expresses 2, 3, 4, 5, 6,7, 8, 9, 10, or more such transport proteins. In some aspects, the cellcan be engineered specifically to express a particular membrane effluxtransport protein, and can be engineered specifically to express two ormore particular membrane efflux transport proteins. Moreover, such cellscan be engineered to express the particular membrane efflux transportproteins at a particular density, or, in polarized cells, at aparticular location on the cell surface, for example, at the basalsurface, at the apical surface, both the basal and apical surfaces, orneither the basal or apical surfaces. Methods for transforming cells toexpress a particular efflux protein transgene are known in the art andare routinely practiced, including those that are described andexemplified herein. Non-limiting examples of membrane efflux transportproteins that are suitable for analysis using the claimed methods anddescribed cells include P-glycoprotein, Multidrug Resistance-AssociatedProtein 2, and Breast Cancer Resistance Protein. Such transporters canhave SEQ ID NOs: 6, 7, or 8, or allelic variants, homologs, and analogsthereof.

The cell is preferably isolated from or alternatively has thecharacteristics of a cell isolated from the gastrointestinal tract ofthe animal. For example, the cell can be isolated from the stomach, thesmall intestine, or the large intestine, including from any subpart ofthese organs. In some preferred embodiments, the cells are intestinalcells, particularly intestinal epithelial cells. In some preferredembodiments, the cells are intestinal cell lines. In highly preferredembodiments, the cells are Caco-2 cells, C2BBe1 cells, HT-29 cells, orT-84 cells. Alternatively, Madin-Darby Canine Kidney (MDCK) cells, acell type known to approximate many of the characteristics of polarizedepithelial cells of the gastrointestinal tract of animals, could also beused as one embodiment of the present invention (Maksymowych, A B andSimpson L L, J. Biol. Chem. 273:21950-57 (1998)). MDCK cells (MDR-MDCK)have already been used to assess human P-gp mediated transport ofblood-brain barrier compounds (Wang, Q. et al., Int. J. Pharmaceutics288:349-59 (2005)).

Modulation of the expression of the at least one membrane effluxtransport protein can occur by any means suitable in the art. In highlypreferred embodiments, the expression of the transport proteins isinhibited. In some aspects, the inhibition is effectuated on the geneticlevel. For example, in cells specifically engineered to express atransgene encoding a particular efflux transport protein, the transgenecan be placed under control of an inducible promoter. Induciblepromoters suitable for use in this invention will be known to those ofskill in the art.

In some preferred embodiments, genes encoding membrane efflux transportproteins such as P-glycoprotein, Multidrug Resistance-Associated Protein2, and Breast Cancer Resistance Protein can be inhibited through the useof a variety of other post-transcriptional gene silencing (RNAsilencing) techniques. RNA silencing involves the processing ofdouble-stranded RNA (dsRNA) into small 21-28 nucleotide fragments by anRNase H-based enzyme (“Dicer” or “Dicer-like”). The cleavage products,which are siRNA (small interfering RNA) or miRNA (micro-RNA) areincorporated into protein effector complexes that regulate geneexpression in a sequence-specific manner.

RNA interference (RNAi) is a mechanism of post-transcriptional genesilencing mediated by double-stranded RNA (dsRNA), which is distinctfrom antisense and ribozyme-based approaches (see Jain K KPharmacogenomics (2004) 5:239-42, for a review of RNAi and siRNA). RNAinterference is useful in a method for inhibiting the expression of amembrane efflux transport protein in an animal such as a human byadministering to the animal a nucleic acid (e.g., dsRNA) that hybridizesunder stringent conditions to a gene encoding a membrane effluxtransport protein, and attenuates expression of the target gene. RNAinterference provides shRNA or siRNA that comprise multiple sequencesthat target one or more regions of the membrane efflux transport proteintarget gene. dsRNA molecules (shRNA or siRNA) are believed to directsequence-specific degradation of mRNA in cells of various types afterfirst undergoing processing by an RNase III-like enzyme called DICER(Bernstein E et al. (2001) Nature 409:363-366) into smaller dsRNAmolecules comprised of two 21 nt strands, each of which has a 5′phosphate group and a 3′ hydroxyl, and includes a 19 nt region preciselycomplementary with the other strand, so that there is a 19 nt duplexregion flanked by 2 nt-3′ overhangs. RNAi is thus mediated by shortinterfering RNAs (siRNA), which typically comprise a double-strandedregion approximately 19 nucleotides in length with 1-2 nucleotide 3′overhangs on each strand, resulting in a total length of betweenapproximately 21 and 23 nucleotides. In mammalian cells, dsRNA longerthan approximately 30 nucleotides typically induces nonspecific mRNAdegradation via the interferon response. However, the presence of siRNAin mammalian cells, rather than inducing the interferon response,results in sequence-specific gene silencing.

Viral vectors or DNA vectors encode short hairpin RNA (shRNA) which areprocessed in the cell cytoplasm to short interfering RNA (siRNA). Ingeneral, a short, interfering RNA (siRNA) comprises an RNA duplex thatis preferably approximately 19 basepairs long and optionally furthercomprises one or two single-stranded overhangs or loops. An siRNA maycomprise two RNA strands hybridized together, or may alternativelycomprise a single RNA strand that includes a self-hybridizing portion.siRNAs may include one or more free strand ends, which may includephosphate and/or hydroxyl groups. siRNAs typically include a portionthat hybridizes under stringent conditions with a target transcript. Onestrand of the siRNA (or, the self-hybridizing portion of the siRNA) istypically precisely complementary with a region of the targettranscript, meaning that the siRNA hybridizes to the target transcriptwithout a single mismatch. In certain embodiments of the invention inwhich perfect complementarity is not achieved, it is generally preferredthat any mismatches be located at or near the siRNA termini.

siRNAs have been shown to downregulate gene expression when transferredinto mammalian cells by such methods as transfection, electroporation,cationic liposome-mediated transfection, or microinjection, or whenexpressed in cells via any of a variety of plasmid-based approaches. RNAinterference using siRNA is reviewed in, e.g., Tuschl T (2002) Nat.Biotechnol. 20:446-8; Yu J-Y et al. (2002) Proc. Natl. Acad. Sci.99:6047-52; Sui G et al. (2002) Proc. Natl. Acad. Sci. USA., 99:5515-20;Paddison P J et al. (2002) Genes and Dev. 16:948-58; Brummelkamp T R etal. (2002) Science 296:550-3, 2002; Miyagashi M et al. (2002) Nat.Biotech. 20:497-500; and, Paul C P et al. (2002) Nat. Biotechnol.20:505-8. As described in these and other references, the siRNA mayconsist of two individual nucleic acid strands or of a single strandwith a self-complementary region capable of forming a hairpin(stem-loop) structure. A number of variations in structure, length,number of mismatches, size of loop, identity of nucleotides inoverhangs, etc., are consistent with effective siRNA-triggered genesilencing. While not wishing to be bound by any theory, it is thoughtthat intracellular processing (e.g., by DICER) of a variety of differentprecursors results in production of siRNA capable of effectivelymediating gene silencing. Generally it is preferred to target exonsrather than introns, and it may also be preferable to select sequencescomplementary to regions within the 3′ portion of the target transcript.Generally it is preferred to select sequences that contain approximatelyequimolar ratio of the different nucleotides and to avoid stretches inwhich a single residue is repeated multiple times.

siRNAs may thus comprise RNA molecules having a double-stranded regionapproximately 19 nucleotides in length with 1-2 nucleotide 3′ overhangson each strand, resulting in a total length of between approximately 21and 23 nucleotides. As used herein, siRNAs also include various RNAstructures that may be processed in vivo to generate such molecules.Such structures include RNA strands containing two complementaryelements that hybridize to one another to form a stem, a loop, andoptionally an overhang, preferably a 3′ overhang. Preferably, the stemis approximately 19 by long, the loop is about 1-20, more preferablyabout 4-10, and most preferably about 6-8 nt long and/or the overhang isabout 1-20, and more preferably about 2-15 nt long. In certainembodiments of the invention the stem is minimally 19 nucleotides inlength and may be up to approximately 29 nucleotides in length. Loops of4 nucleotides or greater are less likely subject to steric constraintsthan are shorter loops and therefore may be preferred. The overhang mayinclude a 5′ phosphate and a 3′ hydroxyl. The overhang may, but need notcomprise a plurality of U residues, e.g., between 1 and 5 U residues.Classical siRNAs as described above trigger degradation of mRNAs towhich they are targeted, thereby also reducing the rate of proteinsynthesis. In addition to siRNAs that act via the classical pathway,certain siRNAs that bind to the 3′ UTR of a template transcript mayinhibit expression of a protein encoded by the template transcript by amechanism related to but distinct from classic RNA interference, e.g.,by reducing translation of the transcript rather than decreasing itsstability. Such RNAs are referred to as microRNAs (miRNAs) and aretypically between approximately 20 and 26 nucleotides in length, e.g.,22 nt in length. It is believed that they are derived from largerprecursors known as small temporal RNAs (stRNAs) or mRNA precursors,which are typically approximately 70 nt long with an approximately 4-15nt loop (Grishok A et al. (2001) Cell 106:23-4; Hutvagner G et al.(2001) Science 293:834-8; Ketting R F et al. (2001) Genes Dev.15:2654-9). Endogenous RNAs of this type have been identified in anumber of organisms including mammals, suggesting that this mechanism ofpost-transcriptional gene silencing may be widespread (Lagos-Quintana Met al. (2001) Science 294:853-8, 2001; Pasquinelli A E (2002) TrendsGen. 18:171-3). MicroRNAs have been shown to block translation of targettranscripts containing target sites in mammalian cells (Zeng Y et al.(2002) Mol. Cell. 9:1327-33).

siRNAs such as naturally occurring or artificial (i.e., designed byhumans) mRNAs that bind within the 3′ UTR (or elsewhere in a targettranscript) and inhibit translation may tolerate a larger number ofmismatches in the siRNA/template duplex, and particularly may toleratemismatches within the central region of the duplex. In fact, there isevidence that some mismatches may be desirable or required as naturallyoccurring stRNAs frequently exhibit such mismatches as do mRNAs thathave been shown to inhibit translation in vitro. For example, whenhybridized with the target transcript such siRNAs frequently include twostretches of perfect complementarity separated by a region of mismatch.A variety of structures are possible. For example, the mRNA may includemultiple areas of nonidentity (mismatch). The areas of nonidentity(mismatch) need not be symmetrical in the sense that both the target andthe mRNA include nonpaired nucleotides. Typically the stretches ofperfect complementarity are at least 5 nucleotides in length, e.g., 6,7, or more nucleotides in length, while the regions of mismatch may be,for example, 1, 2, 3, or 4 nucleotides in length.

Hairpin structures designed to mimic siRNAs and mRNA precursors areprocessed intracellularly into molecules capable of reducing orinhibiting expression of target transcripts (McManus M T et al. (2002)RNA 8:842-50). These hairpin structures, which are based on classicalsiRNAs consisting of two RNA strands forming a 19 by duplex structureare classified as class I or class II hairpins. Class I hairpinsincorporate a loop at the 5′ or 3′ end of the antisense siRNA strand(i.e., the strand complementary to the target transcript whoseinhibition is desired) but are otherwise identical to classical siRNAs.Class II hairpins resemble mRNA precursors in that they include a 19 ntduplex region and a loop at either the 3′ or 5′ end of the antisensestrand of the duplex in addition to one or more nucleotide mismatches inthe stem. These molecules are processed intracellularly into small RNAduplex structures capable of mediating silencing. They appear to exerttheir effects through degradation of the target mRNA rather than throughtranslational repression as is thought to be the case for naturallyoccurring mRNAs and stRNAs.

Thus it is evident that a diverse set of RNA molecules containing duplexstructures is able to mediate silencing through various mechanisms. Forthe purposes of the present invention, any such RNA, one portion ofwhich binds to a target transcript and reduces its expression, whetherby triggering degradation, by inhibiting translation, or by other means,is considered to be an siRNA, and any structure that generates such ansiRNA (i.e., serves as a precursor to the RNA) is useful in the practiceof the present invention.

A further method of RNA interference for use in the present invention isthe use of short hairpin RNAs (shRNA). A plasmid containing a DNAsequence encoding for a particular desired siRNA sequence is deliveredinto a target cell via transfection or virally-mediated infection. Oncein the cell, the DNA sequence is continuously transcribed into RNAmolecules that loop back on themselves and form hairpin structuresthrough intramolecular base pairing. These hairpin structures, onceprocessed by the cell, are equivalent to transfected siRNA molecules andare used by the cell to mediate RNAi of the desired protein. The use ofshRNA has an advantage over siRNA transfection as the former can lead tostable, long-term inhibition of protein expression. Inhibition ofprotein expression by transfected siRNAs is a transient phenomenon thatdoes not occur for times periods longer than several days. In somecases, this may be preferable and desired. In cases where longer periodsof protein inhibition are necessary, shRNA mediated inhibition ispreferable. The use of shRNA is particularly preferred. Typically,siRNA-encoding vectors are constructs comprising a promoter, a sequenceof the target gene to be silenced in the “sense” orientation, a spacer,the antisense of the target gene sequence, and a terminator.

Inhibition of the expression of the membrane efflux transport proteinscan also be effectuated by other means that are known and readilypracticed in the art. For example, antisense nucleic acids can be used.Antisense RNA transcripts have a base sequence complementary to part orall of any other RNA transcript in the same cell. Such transcripts havebeen shown to modulate gene expression through a variety of mechanismsincluding the modulation of RNA splicing, the modulation of RNAtransport and the modulation of the translation of mRNA (Denhardt D T(1992) Ann. N Y Acad. Sci. 660:70-6, 1992; Nellen Wet al. (1993) TrendsBiochem. Sci. 18:419-23; and, Baker B F et al. (1999) Biochim. Biophys.Acta. 1489: 3-18). Accordingly, in certain embodiments of the invention,inhibition of one or more membrane efflux transport proteins in a cellis accomplished by expressing an antisense nucleic acid molecule in thecell.

Antisense nucleic acids are generally single-stranded nucleic acids(DNA, RNA, modified DNA, or modified RNA) complementary to a portion ofa target nucleic acid (e.g., an mRNA transcript) and therefore able tobind to the target to form a duplex. Typically, they areoligonucleotides that range from 15 to 35 nucleotides in length but mayrange from 10 up to approximately 50 nucleotides in length. Bindingtypically reduces or inhibits the function of the target nucleic acid,such as a gene encoding a membrane efflux transport protein. Forexample, antisense oligonucleotides may block transcription when boundto genomic DNA, inhibit translation when bound to mRNA, and/or lead todegradation of the nucleic acid. Inhibition of the expression of amembrane efflux transport protein can be achieved by the administrationof antisense nucleic acids or peptide nucleic acids comprising sequencescomplementary to those of the mRNA that encodes the membrane effluxtransport protein. Antisense technology and its applications are wellknown in the art and are described in Phillips, M. I. (ed.) AntisenseTechnology, Methods Enzymol., 2000, Volumes 313 and 314, Academic Press,San Diego, and references mentioned therein. See also Crooke, S. (ed.)“ANTISENSE DRUG TECHNOLOGY: PRINCIPLES, STRATEGIES, AND APPLICATIONS”(1^(st) Edition) Marcel Dekker; and references cited therein.

Antisense oligonucleotides can be synthesized with a base sequence thatis complementary to a portion of any RNA transcript in the cell.Antisense oligonucleotides can modulate gene expression through avariety of mechanisms including the modulation of RNA splicing, themodulation of RNA transport and the modulation of the translation ofmRNA. Various properties of antisense oligonucleotides includingstability, toxicity, tissue distribution, and cellular uptake andbinding affinity may be altered through chemical modifications including(i) replacement of the phosphodiester backbone (e.g., peptide nucleicacid, phosphorothioate oligonucleotides, and phosphoramidateoligonucleotides), (ii) modification of the sugar base (e.g.,2′-O-propylribose and 2′-methoxyethoxyribose), and (iii) modification ofthe nucleoside (e.g., C-5 propynyl U, C-5 thiazole U, and phenoxazine C)(Wagner R W (1995) Nat. Medicine 1:1116-8; Varga L V et al. (1999)Immun. Lett. 69:217-24; Neilsen P E (1999) Curr. Opin. Biotech. 10:71-5;and, Woolf T M (1990) Nucleic Acids Res. 18:1763-9).

Inhibition of membrane efflux transport proteins can also be effectuatedby use of ribozymes. Certain nucleic acid molecules referred to asribozymes or deoxyribozymes have been shown to catalyze thesequence-specific cleavage of RNA molecules. The cleavage site isdetermined by complementary pairing of nucleotides in the RNA or DNAenzyme with nucleotides in the target RNA. Thus, RNA and DNA enzymes canbe designed to cleave to any RNA molecule, thereby increasing its rateof degradation (Cotten M et al. (1989) EMBO J. 8: 3861-6, 1989; and,Usman N et al. (1996) Curr. Opin. Struct. Biol. 1:527-33).

In preferred aspects of the invention, the cells used in the inventivemethods can be specifically transformed with transcription-silencingnucleic acids such as shRNA or siRNA, or can be transformed with vectorsencoding such nucleic acids such that the cell expresses the inhibitorynucleic acid molecules. Transformation of the cells can be carried outaccording to any means suitable in the art, including those describedand exemplified herein. In specific embodiments, the inhibitory nucleicacid molecules comprise SEQ ID NO: 1, 2, 3, 4, 5, 17, 18, 19, 20, 21,22, 23, 24, 25, or 26, or analogs, homologs, derivatives, or allelicvariants thereof.

In accordance with the inventive methods, test compounds can be screenedat a single dose, or with multiple doses. In some embodiments, the testcompound is evaluated at multiple dosages ranging from the compound'sfree maximal therapeutic plasma concentration (Cmax) to a concentrationequal to or greater than 500-fold over the compound's Cmax. In someembodiment, the test compound is evaluated at multiple dosages rangingfrom the compound's Cmax to a concentration equal to or greater than250-fold over the compound's Cmax. In some embodiments, the testcompound is evaluated at multiple dosages ranging from the compound'sCmax to a concentration equal to or greater than 100-fold over thecompound's Cmax. In some embodiments, the test compound is evaluated atmultiple dosages ranging from the compound's Cmax to a concentrationequal to or greater than 50-fold over the compound's Cmax. In someembodiments, the test compound is evaluated at multiple dosages rangingfrom the compound's Cmax to a concentration equal to or greater than30-fold over the compound's Cmax. In some embodiments, the test compoundis evaluated at multiple dosages ranging from the compound's Cmax to aconcentration equal to or greater than 10-fold over the compound's Cmax.Cmax can be determined according to any means available in the art. Theskilled artisan will appreciate that such means are known and routine inthe art. The compound can be tested at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, or more concentrations within this range.

In some aspects, multiple test compounds are contacted with the cell toevaluate drug-drug interactions. Drug-drug interactions are defined asinfluences of one drug on the pharmacokinetics or pharmacodynamics of asecond drug co-administered to the same subject. Pharmacokinetics refersto the influence of varying drug doses and methods of administration onthe concentration of drug in various body tissues, such as blood, bloodplasma, brain, etc., as a function of time after drug administration.Pharmacodynamics refers to the study of the influence of drug dose androute of administration on the pharmacological response, such as bloodpressure, blood lipid level, number of infectious viral particles in atissue, etc., to drug administration. Drug-drug interactions typicallyoccur by one of three mechanisms: 1) two or more drugs compete for thesame limited quantity of an enzyme or transport protein responsible fortheir metabolism, uptake or efflux in the body; 2) one drug inhibits anenzyme, uptake or efflux transporter that mediates the metabolism,uptake or excretion of one or more other drugs by the body; or 3) onedrug enhances or inhibits the production of an enzyme, uptake or effluxtransporter responsible for the metabolism, uptake or efflux of one ormore other drugs in the body. In some cases the interacting substance isnot a drug, but rather a natural component of a dietary product, such asa component of a fruit juice, such as grapefruit juice, or an herbalsupplement, such as Saint John's wort. Instances of drug-druginteraction associated with a membrane efflux transport protein havebeen reported. For example, the oral bioavailability of digoxin, ap-glycoprotein substrate, was increased by talinolol (Westphal K et al.(2000) Clin. Pharmacol. Ther. 68:6-12). In another instance, renalclearance of digoxin was hampered by clarithromycin resulting inelevated systemic digoxin concentration (Wakasugi H et al. (1998) Clin.Pharmacol. Ther. 64:123-8). Therefore, membrane efflux transport proteinmediated drug-drug interactions may alter the pharmacokinetics of a drugin terms of drug absorption, distribution, and clearance, and may leadto unexpected response to the drug. Cell lines with varying patterns ofefflux transport protein expression offer the ability to identifysubstrates and inhibitors of such protein explicitly, and thus make itpossible to predict potential drug-drug interactions and to provideguidance for adjustment of drug dosage regimen. On Sep. 11, 2006, the USFood and Drug Administration (FDA) announced a draft guidance discussingthe importance of in vitro assays for determining drug-drug interactionsand suggesting ways in which such assays could be carried out. Theguidance also provides recommendations on human clinical studies thatcan be used to confirm or refute the in vitro findings(http://www.fda.gov/cder/drug/drugInteractions/default.htm).

Also featured are methods for screening compounds for gastrointestinalabsorption that utilize a parallel analysis of the inhibition ofdifferent membrane efflux transport proteins in separate cells. Forexample, a given test compound can be screened in a panel of cells, eachcell in the panel being a knockdown for expression of a differentmembrane efflux transport protein, or a different combination ofmembrane efflux transport proteins. Using such methods, those of skillin the art can advantageously determine the contribution of eachindividual transport protein or of particular combinations of transportproteins on the gastrointestinal absorption of a test compound.

Accordingly, in some aspects, the inventive methods comprise inhibitingthe expression of a first membrane efflux transport protein in a firstcell and inhibiting the expression of a second membrane efflux transportprotein in a second cell, contacting the first and second cells with atest compound, and measuring transcellular transport of the testcompound in the first and second cells. The transcellular transportmeasurements from each of the first and second cells can then becompared with reference values for transcellular transport of compoundswith no gastrointestinal absorption, low gastrointestinal absorption,moderate gastrointestinal absorption, or high gastrointestinalabsorption. The measurements indicate the role of the first and secondmembrane efflux transport proteins in transcellular transport of thetest compound. In addition, the measurements relative to the referencevalues are predictive of the gastrointestinal absorption of the compoundin the body of the animal of interest.

In some aspects, the methods comprise inhibiting additional membraneefflux transport proteins such as a third, fourth, fifth, sixth, or moretransport proteins, or combinations thereof, in separate cells, therebyexpanding the number of cells in the panel. Thus, in preferredembodiments, the methods comprise inhibiting the expression of a firstmembrane efflux transport protein in a first cell, inhibiting theexpression of a second membrane efflux transport protein in a secondcell, and inhibiting the expression of a third membrane efflux transportprotein in a third cell, contacting the first, second, and third cellswith a test compound, and measuring transcellular transport of the testcompound in the first, second, and third cells. The transcellulartransport measurements from each of the first, second, and third cellscan then be compared with reference values for transcellular transportof compounds with no gastrointestinal absorption, low gastrointestinalabsorption, moderate gastrointestinal absorption, or highgastrointestinal absorption. The measurements indicate the role of thefirst, second, and third membrane efflux transport proteins intranscellular transport of the test compound. In addition, themeasurements relative to the reference values are predictive of thegastrointestinal absorption of the compound in the body of the animal ofinterest.

Variations on such methods comprise inhibiting the expression of two ormore membrane efflux transport proteins in a first cell, and at leastone membrane efflux transport protein in a second cell. In some aspects,the expression of two or more membrane efflux transport proteins can beinhibited, for example, by means of a single nucleic acid molecule thatcan inhibit the expression of two or more membrane efflux transportproteins. The membrane efflux transport protein in the second cell canbe the same as one of the membrane efflux transport proteins in thefirst cell, or can be a different membrane efflux transport protein.These methods provide the skilled artisan with the advantage of beingable to discern and characterize any synergistic effect of membraneefflux transport proteins in the cell.

The second, third, fourth (and the like) cells can be freshly isolated,established cell lines, or can be cell lines produced de novo. The cellspreferably expresses at least one membrane efflux transport protein,although in some embodiments, the cell expresses 2, 3, 4, 5, 6, 7, 8, 9,10, or more such transport proteins. In some aspects, the cells can beengineered specifically to express a particular membrane effluxtransport protein, and can be engineered specifically to express two ormore particular membrane efflux transport proteins. Moreover, such cellscan be engineered to express the particular membrane efflux transportproteins at a particular density, or, in polarized cells, at aparticular location on the cell surface, for example, at the basalsurface, at the apical surface, both the basal and apical surfaces, orneither the basal or apical surfaces.

Non-limiting examples of membrane efflux transport proteins that arecontemplated for analysis using panels of cells include P-glycoprotein,Multidrug Resistance-Associated Protein 2, and Breast Cancer ResistanceProtein.

The cells are preferably isolated from, or alternatively have thecharacteristics of a cell isolated from the gastrointestinal tract ofthe animal of interest. For example, the cells can be isolated from thestomach, the small intestine, or the large intestine, including from anysubpart of these organs. In some preferred embodiments, the cells areintestinal cells, particularly intestinal epithelial cells. In somepreferred embodiments, the cells are intestinal cell lines, which can beneoplastically transformed, or otherwise immortalized. In highlypreferred embodiments, the cells are Caco-2 cells, C2BBe1 cells, HT-29cells, or T-84 cells.

Transcellular transport of the test compound in any of the first,second, third, or more cells can be measured according to any meanssuitable in the art. Transepithelial electrical resistance (TEER)measurements, which are routinely carried out, can also be used. Liquidchromatography-mass spectrometry (LC-MS) and LC-tandem mass spectrometry(LC-MS-MS) can be used (van Breemen R B, et al. (2005) Expert Opin. DrugMetab. Toxicol. 1:175-85). In addition, fluorescent dyes or radioisotopecan be used, for example, by tagging the test compound with anacceptable dye or isotope as the labels can be conveniently detected byfluorescence or liquid scintillation counting. Non-limiting examplesinclude fluorescent Rhodamine 123, and radiolabeled cyclosporine A,digoxin, ritonavir, taxol, verapamil, and vinblastine (Troutman M T(2003) Pharm. Res. 20:1210-24).

Unidirectional (mucosal-to-serosal transport) or bidirectional(mucosal-to-serosal and serosal-to-mucosal transport) permeability ofthe cells can be measured. In the unidirectional transport, a drugsolution is added to the apical (mucosal) side of the cell monolayers,samples are collected from the basolateral (serosal) side, and apermeability coefficient is determined by the accumulative drugtransported across divided by the time, surface area and doseconcentration. In the bidirectional transport, both permeabilitycoefficients in apical-to-basolateral (mucosal-to-serosal) andbasolateral-to-apical (serosal-to-mucosal) directions are determined.Several mammalian epithelial cell lines plated onto the upper well ofdual well tissue culture plates have been used for the purpose ofstudying the permeability and transport of various chemicals, includingdrugs, toxins and nutrients (Weinstein K et al. in “PharmaceuticalProfiling in Drug Discovery for Lead Selection”, R.T. Borchardt, E. H.Kerns, C. A. Lipinski, D. R. Thakker and B. Wang, eds., pp 217-234, Am.Assoc. Pharm. Scientists, Arlington, Va., 2004. J. Polli and C.Serabjit-Singh, ibid, pp 235-255.)

Measurements of the transcellular transport of the test compound can bedirectly compared with reference values for the transcellular transport,i.e., gastrointestinal absorption rate, efficiency, capacity, etc., ofcompounds in which transcellular transport has been previouslycharacterized. For example, measurements obtained from the test compoundcan be compared to reference values for compounds with nogastrointestinal absorption, with low gastrointestinal absorption, withmoderate gastrointestinal absorption, with high gastrointestinalabsorption, or with any combination of such reference values.Non-limiting examples of such reference values are provided by Table 2(Artursson P. et al. (1991) Biochem. Biophys. Res. Commun 175:880-5).

TABLE 2 Correlation of C2BBe1 permeability and absorption in human cellsP_(app) in C2BBE1 cells % Absorption Drug (10⁻⁶ cm/s) in humanCorticosterone 54.5 100 Testosterone 51.8 100 Propranolol 41.9 90Alprenolol 40.5 93 Warfarin 38.3 98 Metoptolol 27 95 Felodipine 22.7 100Hydrocortisone 21.5 89 Dexamethasone 12.5 100 Salicylic acid 11.9 100Acetylsalicylic acid 2.4 100 Practolol 0.9 100 Terbutaline 0.38 73Atenolol 0.2 50 Mannitol 0.18 16 Arginine-vasopressin 0.14 0Sulphasalazine 0.13 13 1-Deamino-8-D-arginine 0.13 1 Olsalazine 0.11 2Polyethylene glycol 0.052 0

The test compound measurement values experimentally obtained relative tothe reference values is indicative, and at least predictive, of the testcompound's absorption in the gastrointestinal tract of the animal ofinterest. It is contemplated that compounds characterized according tothe methods of the invention can serve as reference compounds,representing the relative degree of gastrointestinal absorption, againstwhich additional test compounds can be compared.

The invention also features methods for inhibiting the expression ofmembrane efflux transport proteins in cells. In some embodiments, themethods comprise stably transforming a cell with a nucleic acid moleculethat interferes with the expression of the membrane efflux transportprotein. The nucleic acid molecule can inhibit the expression byinhibiting the transcription of the gene encoding the membrane effluxtransport protein, or can inhibit the expression by inhibiting thetranslation of mRNA into the protein.

The nucleic acid molecule can be any regulatory gene or fragment of agene whose expression or presence in the cell inhibits transcription ortranslation of the efflux transport protein gene product. In preferredembodiments, the nucleic acid molecule is RNA. In more preferredembodiments, the nucleic acid molecule is interfering RNA, and ispreferably double stranded. Non-limiting examples of interfering RNAinclude siRNA and shRNA.

It has been discovered in accordance with the present invention thatcertain individual nucleic acid molecules can inhibit the expression oftwo or more membrane efflux transport proteins. Accordingly, suchnucleic acid molecules can be advantageously used in any of theinventive methods described and exemplified herein. A non-limitingexample of an individual nucleic acid molecule that can inhibit two ormore membrane efflux transport proteins is SEQ ID NO: 25. This nucleicacid molecule has been demonstrated to inhibit the expression of atleast both BCRP and MRP2. The observation that a single nucleic acidmolecule can inhibit two or more membrane efflux transport proteinsrepresents a significant advance for the determination of the relativecontribution of select membrane efflux transport proteins on cellularabsorption and transport of compounds.

A cell can be transformed with such nucleic acid molecules according toany means available in the art such as those describe or exemplifiedherein. It is preferred that cells are stably transformed with a vectorcomprising a nucleic acid sequence encoding such regulatory nucleic acidmolecules. Any vector suitable for transformation of the particular cellof interest can be used in the present invention. In preferredembodiments, the vector is a viral vector. In more preferredembodiments, the vector is a lentivirus vector.

The regulatory nucleic acid molecule can comprise any sequencecomplementary to, or otherwise amenable to hybridization to and/orinterference with the expression of a gene encoding the membrane effluxtransport protein of interest. Non-limiting examples of such nucleicacid sequences include SEQ ID NOs: 1, 2, 3, 4, 5, 17, 18, 19, 20, 21,22, 23, 24, 25, and 26, and allelic variants thereof. Preferred, butnon-limiting examples of membrane efflux transport proteins includeP-glycoprotein, Multidrug Resistance-Associated Protein-2, and BreastCancer Resistance Protein.

Preferred cells that can be targeted for modulation, particularlyinhibition, of the expression of membrane efflux transport proteins canbe any cell that expresses such transport proteins. Such cells canexpress the transport proteins naturally, or the cells can be engineeredto express the transport proteins. The cells can be isolated fresh froma host organism, or can be cell lines. It is preferred that such cellsbe of a gastrointestinal lineage, and it is particularly preferred thatsuch cells be intestinal epithelial cells. Non-limiting examples of celllines amenable to genetic regulation according to the inventive methodsinclude Caco-2 cells, C2BBe1 cells, HT-29 cells, T-84 cells, and HRT-18cells.

The invention also features isolated nucleic acid molecules for thegenetic regulation of membrane efflux transport expression. Consideredin terms of their sequences, the nucleic acid molecules of the inventionthat encode regulatory, particularly inhibitory, sequences include SEQID NOs: 1, 2, 3, 4, 5, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26, andallelic variants, homologs, and natural mutants of SEQ ID NOs: 1, 2, 3,4, 5, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26. Because such variantsand homologs are expected to possess certain differences in nucleotidesequence, this invention provides isolated polynucleotides that have atleast about 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69% or 70%, more preferably at least about 71%, 72%, 73%, 74%,75%, 76%, 77%. 78%, 79%, or 80%, even more preferably 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, and even more preferably 90%, 91%, 92%,93%, 94%, 95%, and most preferably 96%, 97%, 98% and 99% or moreidentity with any one of SEQ ID NOs: 1, 2, 3, 4, 5, 17, 18, 19, 20, 21,22, 23, 24, 25, and 26. Because of the natural sequence variation likelyto exist among genes encoding these regulatory sequences in differentindividuals, one skilled in the art would expect to find this level ofvariation, while still maintaining the unique properties of thepolynucleotides of the present invention. Accordingly, such variants andhomologs are considered substantially the same as one another and areincluded within the scope of the present invention.

Nucleic acid molecules of the invention may be prepared by two generalmethods: (1) they may be synthesized from appropriate nucleotidetriphosphates, or (2) they may be isolated from biological sources. Bothmethods utilize protocols well known in the art.

The availability of nucleotide sequence information such as the entirenucleic acid sequence of the membrane efflux transport protein, forexample, SEQ ID NOs: 6-8, enables preparation of an isolated nucleicacid molecule of the invention by oligonucleotide synthesis. Syntheticoligonucleotides may be prepared by the phosphoramadite method employedin the Applied Biosystems 38A DNA Synthesizer or similar devices. Theresultant construct may be purified according to methods known in theart, such as high performance liquid chromatography (HPLC). A syntheticDNA molecule so constructed may then be cloned and amplified in anappropriate vector.

Nucleic acids of the present invention may be maintained as DNA in anyconvenient cloning vector. In a preferred embodiment, clones aremaintained in plasmid cloning/expression vector, either of which can bepropagated in a suitable prokaryotic or eukaryotic host cell.

Nucleic acid molecules of the invention include cDNA, genomic DNA, RNA,and fragments thereof which may be single-, double-, or eventriple-stranded. Thus, this invention provides oligonucleotides (senseor antisense strands of DNA or RNA) having sequences capable ofhybridizing with at least one sequence of a nucleic acid molecule of thepresent invention, in particular, SEQ ID NOs: 6-8. Such oligonucleotidesare useful as probes for detecting genes encoding membrane effluxtransport proteins, or for the positive or negative regulation ofexpression of genes encoding a membrane efflux transport protein at orbefore translation of the mRNA into proteins. Methods in whicholigonucleotides or polynucleotides may be utilized as probes for suchassays include, but are not limited to: (1) in situ hybridization; (2)Southern hybridization (3) northern hybridization; and (4) assortedamplification reactions such as polymerase chain reactions (PCR) andligase chain reaction (LCR).

Also featured in accordance with the present invention are vectors andkits for producing transgenic host cells that comprise a polynucleotideencoding a regulatory sequence for inhibiting the expression of amembrane efflux transport protein, or homolog, analog or variant thereofin a sense or antisense orientation, or a construct under control ofcell or tissue-specific promoters and/or other regulatory sequences.Such vectors are suitable for modulating, and preferably inhibiting, theexpression of any membrane efflux transport protein. In preferredembodiments, the membrane efflux transport protein is P-glycoprotein,Multidrug Resistance-Associated Protein 2, or Breast Cancer ResistanceProtein.

Suitable host cells include, but are not limited to, plant cells,bacterial cells, yeast and other fungal cells, insect cells andmammalian cells. More preferred are human cells. Even more preferred arehuman intestinal epithelial cells. Most preferred are Caco-2 cells,C2BBe1 cells, HT-29 cells, or T-84 cells.

Vectors for transforming a wide variety of these host cells are wellknown to those of skill in the art. They include, but are not limitedto, plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterialartificial chromosomes (BACs), yeast artificial chromosomes (YACs), aswell as other bacterial, yeast and viral vectors. In preferred aspectsof the invention, viral vectors are used. It is particularly preferredthat lentiviral vectors are used.

Typically, kits for producing transgenic host cells will contain one ormore appropriate vectors and instructions for producing the transgeniccells using the vector. Kits may further include one or more additionalcomponents, such as culture media for culturing the cells, reagents forperforming transformation of the cells and reagents for testing thetransgenic cells for gene expression or regulation, to name only a few.

In one embodiment, the coding region of the regulatory sequence isplaced under a powerful constitutive promoter, such as the promoters forthe following genes: hypoxanthine phosphoribosyl transferase (HPRT),adenosine deaminase, pyruvate kinase, β-actin, human myosin, humanhemoglobin, human muscle creatine, and others. In addition, many viralpromoters function constitutively in eukaryotic cells and are suitablefor use in the present invention. Such viral promoters include withoutlimitation, Cytomegalovirus (CMV) immediate early promoter, the earlyand late promoters of SV40, the Mouse Mammary Tumor Virus (MMTV)promoter, the long terminal repeats (LTRs) of Maloney leukemia virus,Human Immunodeficiency Virus (HIV), Epstein Barr Virus (EBV), RousSarcoma Virus (RSV), and other retroviruses, and the thymidine kinasepromoter of Herpes Simplex Virus. Other promoters are known to those ofordinary skill in the art. In one embodiment, the coding region of theregulatory sequence is placed under an inducible promoter such as themetallothionein promoter, tetracycline-inducible promoter,doxycycline-inducible promoter, promoters that contain one or moreinterferon-stimulated response elements (ISRE) such as protein kinase R2′,5′-oligoadenylate synthetases, Mx genes, ADAR1, and the like. Othersuitable inducible promoters will be known to those of skill in the art.

The vectors of the invention can be used to transform various cells withthe various regulatory nucleic acid sequences of the invention. Thus,another aspect of the invention features host cells transformed withvectors comprising a nucleic acid sequence encoding a nucleic acidmolecule for modulating, preferably inhibiting, the expression of amembrane efflux transport protein. Numerous techniques are known in theart for the introduction of foreign genes into cells and may be used toconstruct the recombinant cells for purposes of carrying out theinventive methods, in accordance with the various embodiments of theinvention. The technique used should provide for the stable transfer ofthe heterologous gene sequence to the host cell, such that theheterologous gene sequence is heritable and expressible by the cellprogeny, and so that the necessary development and physiologicalfunctions of the recipient cells are not disrupted. Techniques which maybe used include, but are not limited to, chromosome transfer (e.g., cellfusion, chromosome-mediated gene transfer, micro cell-mediated genetransfer), physical methods (e.g., transfection, spheroplast fusion,microinjection, electroporation, liposome carrier), viral vectortransfer (e.g., recombinant DNA viruses, recombinant RNA viruses) andthe like (described in Cline M J (1985) Pharmac. Ther. 29:69-92). It ispreferable that a viral vector be used to transform the cells of theinvention. It is more preferable that the viral vector be a lentiviralvector.

Knockdown cells with inhibited expression of membrane efflux transportproteins can be created by inhibiting the translation of mRNA encodingthe transport protein by “post-transcriptional gene silencing.” The genefrom the species targeted for down-regulation, or a fragment thereof,may be utilized to control the production of the encoded protein.Full-length antisense molecules can be used for this purpose.Alternatively, antisense oligonucleotides targeted to specific regionsof the mRNA that are critical for translation may be utilized. The useof antisense molecules to decrease expression levels of a pre-determinedgene is known in the art. Antisense molecules may be provided in situ bytransforming cells with a DNA construct which, upon transcription,produces the antisense RNA sequences. Such constructs can be designed toproduce full-length or partial antisense sequences. This gene silencingeffect can be enhanced by transgenically over-producing both sense andantisense RNA of the gene coding sequence so that a high amount of dsRNAis produced (for example see Waterhouse et al. (1998) Proc. Natl. Acad.Sci. U.S.A. 95:13959-64). In this regard, dsRNA containing sequencesthat correspond to part or all of at least one intron have been foundparticularly effective. In one embodiment, part or all of the codingsequence antisense strand is expressed by a transgene. In anotherembodiment, hybridizing sense and antisense strands of part or all ofthe coding sequence for one or more membrane efflux transport proteinsare transgenically expressed. Cells of the invention, C2BBE1 cellstransduced with lentiviruses encoding interfering nucleic acid molecules(SEQ ID NOs: 1, 3, 21, 24, 25, and 26), have been placed with the Amer.Type Cult. Coll. (10801 University Blvd., Manassas, Va. 20110-2209) onNov. 6, 2007 and have been assigned Access. Nos. PTA-8752, PTA-8753,PTA-8751, PTA-8754, and PTA-8755, respectively.

The invention also features kits for screening compounds forgastrointestinal absorption in animals. In some embodiments, the kitscomprise a cell that has been transformed with at least one nucleic acidmolecule that inhibits expression of at least one membrane effluxtransport protein, as well as instructions for using the kit in a methodfor screening compounds for gastrointestinal absorption in animals. Thekits of the invention can further comprise a second cell transformedwith a nucleic acid molecule that interferes with the expression of asecond membrane efflux transport protein. The kit of the invention canalso further comprise a second and third cell transformed with a nucleicacid molecule that interferes with the expression of a second and thirdmembrane efflux transport protein.

The cells of the kits can be Caco-2 cells, and preferably are C2BBe1cells. However, any cell that stably expresses at least one membraneefflux transport protein of interest can be used. The cells can betransformed with any nucleic acid molecule that inhibits the expressionof the membrane efflux transport protein of interest, such as but notlimited to, those that are described and exemplified herein. Forexample, the nucleic acid molecule used to transform the cells can haveat least one of SEQ ID NO: 1, 2, 3, 4, 5, 17, 18, 19, 20, 21, 22, 23,24, 25, or 26, or allelic variants thereof.

In the inventive kits, the membrane efflux transport protein of interestwhose expression is inhibited by transformation of the cell with theinterfering nucleic acid can be any efflux transport protein known inthe art, or later discovered. In preferred embodiments, the membraneefflux transport protein is at least one of P-glycoprotein, MultidrugResistance-Associated Protein 2, or Breast Cancer Resistance Protein.

In some embodiments, the inventive kits comprise a cell that expresses amembrane transport efflux protein, a lentivirus vector fortransformation of the cell, and optionally comprises at least onenucleic acid molecule that inhibits the expression of at least onemembrane efflux transport protein expressed by the cell. For example,said nucleic acid molecule can be subcloned into the lentivirus vector,and the vector can be used to transform the cell. It is contemplatedthat any nucleic acid that can inhibit the expression of the membraneefflux transport protein can be subcloned into the lentivirus vector,and used to transform the cell to inhibit the expression of the effluxtransport protein. The kits of this embodiment can comprise instructionsfor using the kit in a method for screening compounds forgastrointestinal absorption in animals. The instructions can also teachhow to subclone an inhibitory nucleic acid into the lentivirus vector.

The invention also features methods for identifying compounds thatinhibit the biological activity of a membrane efflux transport protein.It is preferred that the biological activity that is inhibited is theefflux activity of the transport protein. In some embodiments, themethods comprise inhibiting the expression of a membrane effluxtransport protein in a first cell, contacting the first cell with asubstrate of the membrane efflux transport protein, and determining thebiological activity of the membrane efflux transport protein in thefirst cell. The methods also comprise contacting a second cell in whichexpression of the membrane efflux transport protein is not inhibitedwith a test compound and a substrate of the membrane efflux transportprotein, and in parallel, contacting a second cell in which expressionof the membrane efflux transport protein is not inhibited with asubstrate of the membrane efflux transport protein, and determining thebiological activity of the membrane efflux transport protein in thesecond cell in the presence and absence of the test compound. After thebiological activity of the membrane transport protein is determined forthe first cell, the second cell in the presence of the test compound,and the second cell in the absence of the test compound, the biologicalactivities can be compared. A decrease in the biological activity in thesecond cell in the presence of the test compound relative to thebiological activity in the second cell in the absence of the testcompound, and at least partial identity of the determined value for thebiological activity of the membrane transport protein in the second cellin the presence of the test compound with the determined value for thebiological activity in the first cell is indicative that the testcompound specifically inhibits the membrane efflux transport protein.

Genetic knockdown of the expression of membrane efflux transportproteins inhibits about 20% of the efflux activity of the transportprotein, frequently inhibits about 50% of the activity, and can inhibit80% or more of the activity, as exemplified herein. Thus, by “at leastpartial identity” it is meant that chemical inhibition of a transportprotein by a test compound can match or exceed the levels of inhibitionof efflux activity determined for knockdown cells, or can fall below thelevels of inhibition the efflux activity of knockdown cells. Identitypertains to the inhibition of efflux activity. For illustrationpurposes, genetic inhibition of a first cell may provide for 90%inhibition of efflux activity in the first cell, relative to controls.In comparison, the test compound may provide for 80% inhibition of theefflux activity, relative to controls. Although the test compound inthis scenario provides less inhibition than the genetic knockdown, the80% inhibition provides at least partial identity to the 90% inhibitionof the genetic knockdown. In this example, 90% inhibition by the testcompound would be full or complete identity.

In some embodiments of the inventive methods, inhibiting the expressionof a membrane efflux transport protein in the first cell comprisestransforming the first cell with a nucleic acid molecule that interfereswith the expression of the membrane efflux transport protein. Thenucleic acid molecule can be an interfering nucleic acid molecule asdescribed and exemplified herein. In preferred embodiments, the nucleicacid molecule is RNA, and in more preferred embodiments, the nucleicacid molecule comprises SEQ ID NO: 1, 2, 3, 4, 5, 17, 18, 19, 20, 21,22, 23, 24, 25, or 26 or allelic variants thereof.

In some highly preferred embodiments, the membrane efflux transportprotein of interest is P-glycoprotein, Multidrug Resistance-AssociatedProtein 2, or Breast Cancer Resistance Protein. The P-glycoprotein,Multidrug Resistance-Associated Protein 2, or Breast Cancer ResistanceProtein can be the target of genetic knockdown in the first cell, andthe target of chemical inhibition being screened for in the second cell.In some embodiments, the first cell or second cell is a Caco-2 cell, ora C2BBe1 cell, or combinations thereof.

Any known substrate of the membrane efflux transport protein of interestcan be used in the screening methods. Non-limiting examples of suchsubstrates include digoxin for P-glycoprotein, vinblastine ordinitrophenyl-5-glutathione for Multidrug Resistance-Associated Protein2, and mitoxantrone or estrone-3-sulfate for Breast Cancer ResistanceProtein.

In some aspects, the inventive test compound screening methods can bemodified and adapted to screen for multiple test compounds. For example,two or more membrane efflux transport proteins can be inhibited in thefirst cell, and at least one, preferably two or more test compounds arecontacted with the second cell. The biological activity, such as effluxactivity, of each membrane efflux protein that is inhibited in the firstcell, and that is expressed in the second cell is then determined in thepresence or absence of a test compound, and the determined values of thebiological activity are compared as described above. As such, a decreasein the biological activity in the second cell in the presence of thetest compound(s) relative to the biological activity in the second cellin the absence of the test compound(s), and at least partial identity ofthe determined value for the biological activity of the membranetransport protein(s) in the second cell in the presence of the testcompound(s) with the determined value for the biological activity in thefirst cell is indicative that the test compound(s) specificallyinhibit(s) the membrane efflux transport protein(s).

Compounds identified by any of the foregoing inventive screening methodsare contemplated to be within the scope of this invention. Suchcompounds are preferably inhibitors of membrane efflux transportproteins, and more preferably are inhibitors of P-gp, MRP2, or BCRP.

The following examples are provided to describe the invention in greaterdetail. They are intended to illustrate, not to limit, the invention.

EXAMPLE 1 General Experimental Procedures

Cell Line. The parental cell line, C2BBe1 (ATCC Accession NumberCRL-2102), was used in the experiments described herein to evaluateabsorption potential of candidate drug molecules. The C2BBe1 cell linewas derived from the Caco-2 cell line in 1988 by limiting dilution. Theclone was selected on the basis of morphological homogeneity andexclusive apical villin localization. C2BBe1 cells form a polarizedmonolayer with an apical brush border (BB) morphologically comparable tothat of the human colon. Isolated BBs contain the microvillar proteinsvillin, fimbrin, sucrase-isomaltase, BB myosin-1, and the terminal webproteins fodrin and myosin II. The cells express substantial levels ofBB myosin I similar to that of the human enterocyte. Although clonal,and far more homogenous than the parental Caco-2 cell line with respectto BB expression, these cells are still heterogeneous for microvillarlength, microvillar aggregation, and levels of expression of certain BBproteins.

Cell Seeding and Quality Control. Transwell® devices (Corning, Inc.,Corning, N.Y.) containing monolayers of cells were prepared as follows:Up to one or two dozen 12-well Transwell® devices were prepared at onetime from the same parent stock flask. Any cells not used for seedingTranswell® devices were recultured in T-150 stock tissue culture flasks.Each insert of a 12-well Transwell® device was pretreated with rat tailcollagen to promote cell attachment. Then, 1.5 mL of cell culture media(90% Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovineserum) was added to the bottom wells of a 12-well Transwell® device. Thecells were detached from T-150 tissue culture flasks by trypsinization,and resuspended in cell culture media. Clumps of cells were broken up byrepeated pipetting to generate a uniform suspension of cells. The numberof cells in suspension was counted using a hemocytometer. Supplementalcell culture medium was added to the cell suspension to bring the cellcount to approximately 136,000 cells per mL. 0.5 mL of cell suspension,containing approximately 68,000 cells, was added to each upper well ofthe 12-well Transwell® device. The cells were allowed to attachovernight and fed with fresh cell culture medium the following day byadding 0.5 mL of medium to the upper chamber and 1.5 mL of medium to thelower chamber of each well. Medium was changed every other day for atleast 20 days prior to testing a randomly selected device from each lotfor cell confluence and transporter function.

Quality control (QC) testing of a batch of Transwell® devices wascarried out as follows: Randomly selected inserts from a batch ofTranswell® devices were removed for the QC assay. The cells were placedin a blank Transwell® bottom plate containing Hank's Balanced SaltSolution (HBSS) pH 7.4 containing 10 mM HEPES and 15 mM glucose (HBSSg)pre-warmed to 37° C. The medium was aspirated from the wells and HBSSgwas used to rinse the cell monolayers on the inserts. Fresh HBSSg wasadded after the monolayers were washed, the inserts were removed fromthe assay plate, and the transepithelial electrical resistance (TEER)value of the monolayers was determined using an ENDOHM transepithelialelectrical resistance measurement apparatus (World PrecisionInstruments).

Monolayers having a TEER value above 100 ohm/cm² are consideredacceptable for use in permeability studies. If the TEER value fell belowthis range, the rest of the batch was re-cultured with fresh medium foradditional time and retested. If the batch failed three times, theentire batch was rejected. Sample wells from the batch that were abovethe acceptable limit were tested for permeability of reference compoundsas follows: The following pre-warmed QC solution was added to the upperchamber of the Transwell® insert; 0.5 mM Lucifer Yellow, 10 μM Atenolol,10 μM Propranolol, 10 μM Pindolol and 10 μM Digoxin in HBSSg pH 7.4±0.2.The bottom wells contained HBSSg pre-warmed to 37° C. The Transwelldevice was placed in a humidified incubator and incubated for 2 hours at37° C. in an atmosphere containing 5% CO₂. At end of the incubationperiod, samples were withdrawn from the bottom well for analysis ofLucifer Yellow content by fluorescence detection, and the othercompounds by LC/MS/MS.

Permeability values were calculated from the donor (upper well)concentrations and net increases in receiver (bottom well)concentrations at the 2 hour sampling interval. The permeability for thetime interval, Papp (t1−t2), was calculated according to the followingformula: Papp(t1−t2)=((Ct2−Ct1)/(t2−t1))×Vr/(A×Cd). Ct2−Ct1 is thecumulative concentration difference in the receiver (bottom) compartmentat each time interval in μM, (in this example Ct1 is assumed to be “0”because the entire time interval (120 min) is used in the calculation);Vr is the volume of the receiver compartment (in cm³); A is the area ofthe cell monolayer (1.13 cm² for 12-well Transwell®), and Cd isconcentration in the donor sample compartment (upper well) in μM, whichis equal to the concentration in the donor solution described above. Theapparent permeability for each compound used for QC purposes was theaverage of all Papp values calculated for all replicates tested,typically 3 replicate inserts per assay condition.

This assay was repeated by adding the QC solution to the bottom well ofthe Transwell® and measuring the digoxin concentration in the upper wellafter 2 hours. A calculated Papp for digoxin transport from the lower tothe upper well that is at least 3 times higher than that calculated fortransport from the upper to the lower well indicates functionalexpression of P-gp in the cell monolayers.

shRNA. To knockdown P-gp expression and activity in the C2BBe1 parentalcell line, RNAi technology was used. The goal was long term silencing ofthe P-gp gene. Five 21 nucleotide shRNA duplexes (SEQ ID NOs: 1-5) fromfive different parts of the human P-gp genome (Gen Bank Accession No.NM_(—)000927) were designed using the MISSION® search database of theSigma-Aldrich™ website, which is produced and distributed under licensefrom the Massachusetts Institute of Technology.

Calcein-AM assay. To determine activity of the P-gp protein, calcein-AMassays were conducted. Calcein-AM(3′,6′-Di(O-acetyl-2′,7′-bis[N,N-bis(carboxymethyl)-aminomethyl]fluoresceintetraacetoxymethyl ester) is a hydrophobic ester of the fluorescentmolecule calcein. Calcein-AM is converted to the fluorescent parentcalcein by intracellular sterases. Calcein-AM is also a substrate ofP-gp (U.S. Pat. No. 5,872,014). Calcein-AM cannot readily enter cellswhen P-gp is present in the cell membrane and functional. However, whenP-gp is not present or not functional, calcein-AM can readily enter acell and be quickly converted to its fluorescent counterpart, calcein.Therefore, in the calcein-AM assay, high intracellular fluorescence isan indication of low P-gp expression or function.

To determine the P-gp activity, cells were plated in 96-well tissueculture plates and cultured for 48. After 48 hours, cell culture mediawas removed and the cells were washed with phosphate buffered salinesolution (PBS). After washing, cells were incubated with 1 μM calcein-AMfor 30 minutes. Parental cells treated with Cyclosporin A, anestablished P-gp inhibitor, were used as a positive control to verifythe P-gp inhibition increased the intracellular fluorescence in parentalcell lines. After incubation with calcein-AM, the cells were rinsed withfresh control buffer and fluorescence was measured using a Fluo-starfluorescence plate reader (BMG Lab Technologies, NC) with excitation andemission filters set at wavelengths of 485 nm and 538 nm, respectively.

RT-PCR. Total RNA was extracted from cells according to the followingprotocol. Cells were harvested from culture by centrifugation,resuspended in 1 ml of TRIzol® reagent (Invitrogen, Carlsbad, Calif.),and incubated at room temperature for 5 minutes with gentle shaking. Thesuspension was transferred to a 1.5 ml centrifuge tube, to which 200 μlof Chloroform was added, followed by an additional 5 minute incubationperiod with shaking. After incubation, the suspension was centrifuged at12,000×g for 12 minutes at 4° C. The supernatant was transferred to afresh, sterile 1.5 ml centrifuge tube, and supplemented with 0.5 ml of2-propanol per 1 ml of TRIzol reagent, and incubated for 10 minutes atroom temperature. The mixture was then centrifuged at 12,000×g for anadditional 12 minutes at 4° C. After centrifugation, the supernatant wasremoved and the pellet was washed with 75% ethanol. The sample wasre-centrifuged at 7500×g for 5 minutes at 4° C., followed by the removalof the supernatant. The pellet was allowed to air dry at roomtemperature. The pellet was resuspended in pre-warmed (55° C.)nuclease-free water, and incubated at 55° C. for 10 minutes. RNA yieldand purity were quantified by absorbance at 260 nm and 280 nm.

RT-PCR mixtures were set up as 50 μl samples containing buffer, 1-2 μgof RNA, 10 μM of Forward primer, 10 μM of Reverse primer, Taqpolymerase, and water. Primers to amplify human P-gylcoprotein were asfollows: P-gp Forward 5′-GCTCCTGACTATGCCAAAGC-3′ (SEQ ID NO: 9); and,P-gp Reverse 5′-TCTTCACCTCCAGGCTCAGT-3′ (SEQ ID NO: 10). Primers toamplify human MRP2 were as follows: MRP2 Forward5′-CTGGTTGGGAACCTGACTGT-3′ (SEQ ID NO: 11); and, MRP2 Reverse5′-CAACAGCCACAATGTTGGTC-3′ (SEQ ID NO: 12). Primes to amplify human BCRPwere as follows: BCRP Forward 5′-GTGGCCTTGGCTTGTATGAT 3′ (SEQ ID NO:13); and, BCRP Reverse 5′-GATGGCAAGGGAACAGAAAA 3′ (SEQ ID NO: 14). Humanβ-actin was amplified in parallel as a positive control using thefollowing primers: β-actin Forward 5′-ACTATCGGCAATGAGCGGTTC-3′ (SEQ IDNO: 15); and, β-actin Reverse 5′-AGAGCCACCAATCCACACAGA-3′ (SEQ ID NO:16).

The PCR cycles proceeded in a programmable thermocycler with thefollowing parameters: cDNA synthesis, 1 cycle, 55° C. for 30 minutes;denaturation, 1 cycle, 94° C. for 2 minutes; PCR amplification, 40cycles of 94° C. for 15 seconds, 62° C. for 30 seconds, 72° C. for 1minute; and, final extension, 1 cycle, 72° C. for 5 minutes.Amplification was confirmed by electrophoresis in 2% agarose gels.

Western Blotting. Cellular monolayers were grown to confluency in 90%DMEM+10% FBS, media was removed, and monolayers were washed using 1×PBS.To lyse the cells, 500 μl of RIPA lysis buffer, 1× (Santa CruxBiotechnologies, CA, Cat #sc-24948), was applied to the cells andincubated on ice for 10 min. Lysed cells were harvested and centrifugedat 12,000×g 4° C. The supernatant/protein lysate was transferred to aclean tube, and the protein concentration was determined following theprotocol of the BCA protein assay kit (Pierce, Ill., Cat #23225).

Protein extracts were subject to SDS PAGE as follows. About 25 to 50 μgof protein sample was loaded onto 8% SDS polyacrylamide gels, and run at130 volts for 1 hour. Following electrophoresis, proteins weretransferred to PVDF membranes using a BioRad MiniProtean 3electrophoresis cell following a protocol provided by Bio-Rad.

After the proteins were transferred, the membranes were blocked with0.2% Iblock solution (Applied Biosystems, CA. catalog #T2015) inTris-Buffered Saline (TBS) containing 0.05% Tween-20™ (TBST) for 1 hour.Blocked membranes were incubated with primary antibody; for P-gp mouseanti-human P-gp, C494, Abcam, Cambridge, Mass., catalog# ab2265 diluted1:500 in blocking solution overnight at 4° C.; for MRP2 polyclonalrabbit anti-human MRP2 antibody (Abcam, Cambridge, Mass., Cat# 50213)diluted 1:1,000 and; for BCRP mouse anti-human BCRP antibody (Abcam,Cambridge, Mass., Cat# ab3380). Unbound antibody was removed by washing3× in TBST. After washing, the membranes were reacted with the secondaryantibody, goat anti-mouse IgG linked to horse radish peroxidase, (goatanti-mouse IgG-HRP, Chemicon catalog #AP124) diluted 1:25,000 in blocksolution for 1 hour. Unbound secondary antibody was removed by washing 3times with TBST. Bound antibody was visualized using Super Signal WestFemto Chemiluminescent Substrate (Pierce, catalog #34094), and viewedwith Epi Chem II darkroom from UVP, Inc (Upland, Calif.) following themanufacturer's protocols.

Bidirectional Transcellular Transport assay. Cell monolayers were grownto confluence (approximately 20 to 28 days after seeding) oncollagen-coated, microporous, polycarbonate membranes in 12-well CostarTranswell® plates containing 90% DMEM+10% FBS in the top and bottomwells. Growth medium was changed every two to three days. Spent mediumwas removed by aspiration, and fresh medium was added to each of thewells. All wells were given fresh medium the day prior to the transportassays. Plates were certified as meeting in-house acceptance criteriaprior to studies with test compounds. Details of the plates used andtheir certification are shown in the examples below. Plate certificationusing reference compounds was performed using Hank's Balanced SaltSolution containing 10 mM HEPES and 15 mM glucose at a pH of 7.4±0.1.

The permeability assay buffer for the test articles was Hank's BalancedSalt Solution containing 10 mM HEPES, 15 mM glucose, at a pH of 7.4±0.1.The dosing solution concentrations in assay buffer varied with the testcompound. Typical concentrations of test compound were 10 μM in Hank'sBalanced Salt Solution containing 10 mM HEPES, 15 mM glucose, at a pH of7.4±0.1. At each time point, 1 and 2 hours, a 200-μL aliquot was takenfrom the receiver chamber (the bottom or basolateral chamber for apicalto basolateral [A-to-B] permeability determinations or the top or apicalchamber for basolateral to apical permeability [B-to-A] determinations)and replaced with fresh assay buffer. Cells were dosed on the apicalside (A-to-B) or basolateral side (B-to-A) and incubated at 37° C. with5% CO₂ and 90% relative humidity. Each determination was performed induplicate. The permeability of Lucifer Yellow, a monolayer integritymarker compound, was also measured for each monolayer using afluorescence assay. Lucifer Yellow Papp values are examined to determinewhether monolayer integrity was impaired during the permeation study.Monolayers exhibiting abnormally high Lucifer Yellow permeability valueswere excluded from further analysis. All other compounds were assayed byLC/MS using electrospray ionization as summarized below.

The apparent permeability, Papp, and percent recovery were calculatedaccording to the following formulas:Papp=(dCr/dt)×Vr/(A×Cd0)Percent Recovery=100×(((Vr×Crfinal)+(Vd×Cdfinal))/(Vd×Cd0))where, dCr/dt is cumulative concentration in the receiver compartmentversus time; Vr is the volume of the receiver compartment in cm³; Vd isthe volume of the donor compartment in cm³; A is the area of the cellmonolayer (1.13 cm² for 12-well Transwell); Cd0 is the concentration ofthe dosing solution at time 0; Crfinal is the cumulative receiverconcentration at the end of the incubation period; and, Cdfinal is theconcentration of the donor at the end of the incubation period.

Summary of LC/MS Analytical Methods. A liquid chromatography instrument(LC) capable of generating a gradient of eluting buffer (mobile) phasewas used. A chromatography column (Keystone Hypersil BDS C18 30×2.0 mmi.d., 3 μm, with guard column) was connected to the LC, and a 10 μLsample of buffer from the transport assay was injected into the columnby the autosampler connected to the LC. Two mobile phases werecontinuously mixed in various proportions to establish a compositionalgradient. Typical mobile phases used for this assay are an aqueousbuffer, such as 25 mM Ammonium Formate Buffer, pH 3.5, and an organicsolvent, such as acetonitrile. The elution gradient was formed by mixingappropriate proportions of mobile phases from two mobile phasereservoirs. In the example listed below, one reservoir contained theaqueous buffer and the second reservoir contained a mixture ofacetonitrile and aqueous buffer in the proportion of 9:1(volume:volume). The gradient program in the LC can be set to form avariety of gradients from linear, in which the composition changes frombuffer to acetonitrile plus buffer at a fixed rate, to ballistic, inwhich the composition changes suddenly from buffer to acetonitrile plusbuffer at a specific time in the analysis. Gradient program conditionsfor the analysis used herein are listed in Table 3, in which % A refersto the fraction of aqueous buffer in the gradient and % B refers to thefraction of acetonitrile-buffer mixture in the gradient. The time columnrefers to the time after the sample is injected with 0.0 minutes beingthe sample injection point. In this example the gradient is a ballisticgradient, suddenly changing composition at 1.5 minutes after sampleinjection.

TABLE 3 LC gradient program conditions Time (Min) % A % B 0.0 100 0 0.5100 0 1.5 0 100 2.0 0 100 2.1 100 0 3.5 100 0

The LC autosampler syringe was rinsed with 0.2% formic acid inwater/acetonitrile/2-propanol: 1/1/1 (v/v/v) between injections.

The eluant from the chromatographic column was directed to theelectrospray interface of a triple quadropole mass spectrometer (MS/MS),where the solvent and buffer were evaporated, and compounds eluted fromthe chromatographic column were ionized to form positive or negativeions.

In the examples below an instrument, typically a PE SCIEX API 2000, 3000or in some cases a 4000 model, was used to separate and detect the ions.Triple quadropole instruments, such as these, can separate parent ionsusing the first quadropole magnetic, fragment them in the secondquadropole chamber and detect specific fragments of the parent ion usingthe third quadropole to focus ions of a pre-specified mass onto theinstrument's detector. This mode of detection is frequently referred toas Multiple Reaction Monitoring or MRM. MRM permits very specific andsensitive detection of compounds of interest with mass resolutions of atleast ±1 atomic mass units and limits of detection in the nanogram permilliliter range.

Typical parent and fragment ions used for detection of the compoundsmentioned in the examples are presented in Table 4.

TABLE 4 Parent and fragment ions used for detection Compound Q1/Q3Atenolol +267.4/145.2 Propanolol +260.4/116.2 Pindolol +249.3/116.2Digoxin +798.6/651.5

Q1 refers to the mass selection setting of the first quadropole magnetand Q3 refers to the mass selection setting of the second quadropolemagnet. The “+” sign refers to the sign of the charge on the ions beingmonitored.

Another parameter that can be adjusted on these mass spectrometers isthe dwell time, which refers to the time period in which the twoquadropoles are set to select and detect a particular combination ofparent and fragment (daughter) ions. Multiple compounds can be detectedin the same chromatographic analysis by appropriate adjustment of thechromatographic conditions and the mass spectrometer dwell times.Typical dwell times range from about 10 to about 100 milliseconds perion pair combination. Skilled analysts can usually determine acombination of chromatographic conditions and dwell times that willallow detection and quantification of up to about 6 compounds in thesame sample, provided that their ion masses differ by at least 5 atomicmass units.

Analytical standards with concentrations ranging from about 1 ng/mL upto about 1,000 ng/mL were prepared in the same matrix as used fortransport assay samples. A standard curve was prepared by plotting theMS/MS detector response versus the standard concentration. The standardcurve was fitted to a linear or polynomial response curve using softwareprovided by the instrument manufacturer. The concentration of compoundin the unknowns was determined by back calculating from the detectorresponse. Alternatively, the ratio of detector responses between thecompounds of interest and a reference standard compound added to thestandards and samples at a fixed concentration is used to construct thestandard curve and quantify unknowns. This is known as the internalstandard method of sample quantification.

EXAMPLE 2 Transduction of C2BBe1 Cells with Interfering Nucleic AcidSequences

Interfering nucleic acid sequences were subcloned into MISSION™ shRNAHuman Tumor Suppressor Lentiviral Transduction Particles (Sigma-Aldrich,St. Louis, Mo.). Lentiviruses containing the interfering sequences (SEQID NOs 1, 2, 3, 4, or 5) were used to transduce C2BBe1 cells accordingto the manufacturer's protocols. In brief, C2BBe1 cells were seeded intoa 96 well plate at 1.6×10⁴ cells per well in cell culture media (90%DMEM+10% FBS), and incubated at 37° C. for 24 hours prior totransduction. On the day of transduction, media was removed from thewells and lentiviral particles were added to the wells at 0.5, 1.0, or5.0 MOI (multiplicity of infection) and incubated with the cells for 24hours at 37° C. Media containing unbound lentiviral particles wasremoved after this incubation, and fresh media was supplied. At 48 hourspost-transduction, selective medium containing 10 μg/mL puromycin wasadded and changed every 3-4 days thereafter to select for transducedcells. Five puromycin-resistant isolates generated at an MOI of 1.0 wereprepared as individual cell clones, giving rise to shRNA/P-gp clones#83, 84, 85, 86, and 87 (these clones correspond to transduction withlentiviruses containing SEQ ID NOs 1, 2, 3, 4, or 5, respectively). Eachof the five cell clones were expanded and evaluated using the variousassays described herein. shRNA/P-gp clones #83 and 85 have been placedwith the Amer. Type Cult. Coll. (10801 University Blvd., Manassas, Va.20110-2209) on Nov. 6, 2007 and have been assigned Access. No. PTA-8752and Access. No. PTA-8753, respectively.

EXAMPLE 3 P-gp Gene Expression in shRNA/P-gp Clone Cells

Gene expression of P-gp in knockdown cells (shRNA/P-gp clones #83, 84,85, 86, and 87) was determined by observing P-gp mRNA content, proteincontent and protein functional activity. RT-PCR was used to determinethe expression of the P-gp mRNA. Total cellular RNA was harvested andamplified using primers specific to P-gp mRNA. RT-PCR products were thenseparated by electrophoresis on a 2% agarose gel. It was determined that4 of the 5 shRNA/P-gp clone cells contained substantially less P-gp mRNAthan control C2BBe1 cells (FIG. 2, top panel). β-actin was amplified, inparallel, as a positive control, and its expression was not affected bythe interfering shRNA (FIG. 2, bottom panel).

The PCR result for clone 84 is confounded by the apparent presence ofhigh amounts of primer dimmer in that gel lane. Calcein-AM assaysindicate that P-gp function is knocked down in all 5 clones (FIG. 1).Functional assays measuring calcein-AM uptake indicate that theknockdown of P-gp mRNA occurred at around 80% to 90% efficiency for sometransducants (FIG. 1).

A bidirectional transport experiment was carried out to determine theefflux ratio of digoxin, a known P-gp substrate, on shRNA/P-gp clonecells. The results are shown in FIG. 3. Each of the shRNA/P-gp clonecells demonstrated an increase in apical to basal flux, and furtherdemonstrated that the efflux ratio of Papp (B-A/A-B) was significantlylower than for un-knockdown control cells (Un-KD). The results confirmthat P-gp efflux activity was inhibited by shRNAs targeted to the P-gpgene.

To further evaluate P-gp gene expression in shRNA/P-gp clone cells,protein was obtained from cell lysates, quantified, and separated on an8% Precise® SDS-PAGE protein gel. After separation, proteins weretransferred to a PVDF membrane and evaluated by immunoblotting asdescribed in Example 1. After immunoblotting was complete, membraneswere exposed to a chemiluminescent horseradish peroxidase substrate andprotein content was visualized using a luminometer. As shown in FIG. 4,Western blot analysis indicated a substantial decrease in the amount ofP-gp present in shRNA/P-gp clone cells, as compared to the controlcells. Table 5 provides the optical density for each of the P-gp andβ-actin bands on the Western blot shown in FIG. 4. The ratio of opticaldensities for P-gp and β-actin were calculated and used to determinepercent knockdown. A graphical representation of the percent inhibitionis provided in FIG. 5.

TABLE 5 Optical density for Western blot of knockdown P-gp expression inshRNA/P-gp C2BBe1 clone cells. Calibrate Calibrate Value Value Ratio ofSample ID P-gp Actin P-gp/Actin % of inhibition shRNA/P-gp #83 1309 14740.89 37.71 shRNA/P-gp #84 1240 1712 0.72 49.19 shRNA/P-gp #85 1195 17160.70 51.11 shRNA/P-gp #86 1052 1369 0.77 46.06 shRNA/P-gp #87 967 10370.93 34.58 C2BBe1 wild type 958 672 1.42

P-gp protein activity in shRNA/P-gp clone cells was determined using acalcein-AM assay as previously described in Example 1. Results are shownin FIG. 1. The results demonstrate that calcein fluorescence issubstantially increased in all of the shRNA/P-gp genetic knockdown cells(KD), relative to the unknockdown control cells (Un-KD). Each shRNAchosen was able to significantly inhibit P-gp activity in the cells. Incontrast with the results of the RT-PCR analysis, the calcein-AM assayresults show that P-gp knockdown efficiency ranged from about 70% toabout 90% among the different shRNAs. In parallel, shRNA/P-gp clonecells were also treated with a known chemical inhibitor of P-gpactivity, cyclosporin A (CsA). In each of the different shRNA/P-gp clonecells tested, a small enhancement of calcein fluorescence, correspondingto an increase in P-gp inhibition, was observed upon treatment with CsA.Although none of the increases in fluorescence was determined to bestatistically significant (Student's “t” test) over the geneticknockdown alone (FIG. 1), these results suggest that some residual P-gpactivity remains.

EXAMPLE 4 Inhibition of MRP2 Expression in C2BBe1 Cells

For this example, gene expression of MRP2 in C2BBe1 cells was determinedby observing both the MRP2 mRNA content as well as the protein contentof the cells following viral transduction. To examine mRNA content,total cellular RNA was harvested and amplified by RT-PCR using primersspecific to the MRP2 gene (SEQ ID NO: 11 and 12). The RT-PCR productswere then analyzed after separation by electrophoresis on a 2% agarosegel.

To further evaluate MRP2 gene expression in MRP2 knockdown cells, celllysates were obtained, their protein content quantified, and individualproteins separated on an 8% Precise® SDS-PAGE protein-separation gel asdescribed in Example 1. After separation, proteins were transferred to aPVDF membrane and MRP2 was visualized by immunoblotting using 1:1000dilution of rabbit anti-MRP2 (Abcam, Cambridge Mass., Cat# ab50213,Lot#351245). After immunoblotting, membranes were exposed to achemiluminescent horseradish peroxidase substrate and band stainingintensity was visualized using a luminometer. A decrease in bandstaining intensity by Western blot analysis would indicate a decrease inthe amount of MRP2 protein present in MRP2 knockdown cells as comparedto vector control cells.

Knockdown of MRP2 by Interfering RNA:

To target knockdown expression of MRP2, C2BBe1 cells were transducedwith lentiviruses containing nucleic acid inserts SEQ ID NOs 17, 18, 19,20, or 21, which encode interfering RNA. Our experiments indicated thatshRNA targeted to MRP2 (shRNA/MRP2) required higher MOIs and longerincubation times with the lentivirus than shRNA targeted to BCRP(shRNA/BCRP) and P-gp (shRNA/P-gp) in order to achieve measurable MRP2knockdown. After testing MOIs (multiplicity of infection) from 0.5 to10, RT-PCR analysis showed that an MOI of 10 is optimal forhigh-efficiency MRP2 knockdown. The incubation time of shRNA/MRP2 viralparticles also was extended to 2 days in the transduction experiments.All other experimental conditions were the same as described in Example2.

Five puromycin-resistant isolates were prepared as individual cellclones, giving rise to shRNA/MRP2 clones #3, 4, 5, 6, and 7 (theseclones correspond to transduction with lentiviruses containing SEQ IDNOs 17, 18, 19, 20, or 21, respectively). Each of the five cell cloneswere expanded and evaluated using the various assays described herein.shRNA/MRP2 clone #7 has been placed with the Amer. Type Cult. Coll.(10801 University Blvd., Manassas, Va. 20110-2209) on Nov. 6, 2007 andhas been assigned Access. No. PTA-8751.

MRP2 Knockdown Results:

1.1 Expression of mRNA of MRP2 in Knockdown Clone Cells.

FIG. 6 shows expression of MRP2 mRNA in C2BBe1 cells following viraltransduction with shRNA/MRP2 sequences (SEQ ID NOs 17, 18, 19, 20, or21, respectively). Vector control (VC) cells were prepared from cellstransducted with lentiviruses encoding a non-interfering nucleic acidsequence (SEQ ID NO 27), as described in Example 7. Methods weredescribed in Example 1. Total cellular RNA from cells with passagenumbers ranging from 2 to 5 was isolated after 5 to 7 days of growth.Parallel RT-PCR results showed inhibition of expression of MRP2 mRNA inshRNA/MRP2 clones #3, #4 and #7 (FIG. 6). Analysis of the RT-PCR resultsbased on the ratio of MRP2 mRNA band intensity to β-actin mRNA bandintensity (see Table 6) indicated MRP2 mRNA expression was decreasedfrom 15% to 19% by genetic MRP2 knockdown.

TABLE 6 Percent knockdown of MRP2 mRNA expression in C2BBe1 cellstransduced with lentiviruses containing shRNA/MRP2 inserts. PercentRatio of MRP2 band intensity decrease in ratio C2BBe1cell to β-actinband intensity compared to control Vector Control 0.67 — shRNA/MRP2 #30.57 15.12 shRNA/MRP2 #4 0.57 15.31 shRNA/MRP2 #5 0.63 6.04 shRNA/MRP2#6 0.72 −7.59 shRNA/MRP2 #7 0.54 19.53

1.2 Expression of Protein of MRP2 in shRNA/MRP2 Clone Cells.

Cell lysate content of MRP2 protein from shRNA/MRP2 clones cells andvector control cells was determined by Western blot analysis asdescribed in Example 2. As seen in FIG. 7, Western blot analysisindicated a reduction of MRP2 protein in shRNA/MRP2 clones #4, 5, 6, and7 when compared to vector control (VC) cells. Based on RT-PCR andWestern blot results, C2BBe1 cells transduced with lentivirusescontaining nucleic acid inserts corresponding to SEQ ID NO 21(shRNA/MRP2 clone #7) showed the greatest degree of MRP2 knockdown ofthe 5 interfering sequences examined.

Functional knockdown of MRP2 will be assessed by measuring thebidirectional transport of one or more of the following compounds, knownto be MRP2 substrates, across cell monolayers plated on Transwell®devices as described in Example 1: BCECF(2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein);Bilirubin-diglucuronide; BQ123 (cyclic pentapeptide, endothelin receptorantagonist); Chrysin; CPT11 (irinotecan); Furosemide; Genistin;Glutathione-S—S-glutathione; Glutathion-methylfluorescein;Grepafloxacin; Methotrexate; Nethylmaleimide-S-glutathione; SN 38 (CPT11active metabolite); Sulfotaurolithocholic acid (STLC); Telmisaltan (BIBR277) and its glucuronide metabolite; Temocaprilat; Vinblastine;Cisplatin or other established MRP2 substrates which show efflux acrossparental cell monolayers.

EXAMPLE 5 Inhibition of BCRP mRNA Expression and Activity in C2BBe1Cells

1. Molecular Biological Characterization of BCRP mRNA Expression in BCRPKnockdown Cells

Gene expression of BCRP in knockdown cells was determined by observingBCRP mRNA content, protein content and protein functional activity. Theparental cell line, C2BBe1 (ATCC Accession Number CRL-2102), was used toevaluate genetic inhibition of BCRP expression by transformation ofcells with lentiviral vectors encoding interfering RNA.

Five 21-nucleotide sequences (SEQ ID NOs: 22-26) targeting fivedifferent parts of the human BCRP gene (GeneBank Accession No.NM_(—)004827) were designed using the MISSION® search database of theSigma-Aldrich™ website, which is produced and distributed under alicense from the Massachusetts Institute of Technology. Interferingnucleotide sequences were subcloned into MISSION® shRNA LentiviralTransduction Particles (Sigma-Aldrich, St. Louis, Mo.), and used totransform C2BBe1 cells according to the general procedures set forth inExamples 1 and 2. Five stable shRNA/BCRP transformants, produced bytransduction, at an MOI of 1.0, with lentiviruses containing SEQ ID NOs:22, 23, 24, 25, or 26, were cloned, giving rise to shRNA/BCRP clones#798, 799, 800, 801, and 802, respectively. shRNA/BCRP clones #800 and801 have been placed with the Amer. Type Cult. Coll. (10801 UniversityBlvd., Manassas, Va. 20110-2209) on Nov. 6, 2007 and have been assignedAccess. Nos. PTA-8754 and PTA-8755, respectively.

ShRNA/BCRP clones were evaluated for knockdown expression of the BCRPgene. Expression of BCRP was determined by observing BCRP mRNA contentby RT-PCR amplification as described above, as well as by observing theprotein content as measured by Western blotting.

For RT-PCR, total cellular RNA was harvested as described in Examples 1and 3 and amplified by RT-PCR using primers specific to BCRP mRNA (SEQID NOs: 13 and 14). The RT-PCR products were separated byelectrophoresis on a 2% agarose gel and analyzed to determine whethershRNA/BCRP clone cells produced substantially less BCRP mRNA than C2BBe1cells transduced with a non-interfering shRNA vector control. As shownin FIGS. 8 and 9, all five nucleic acid inserts specific for BCRPsignificantly reduced BCRP mRNA expression relative to the levelexpression observed for shRNA vector control C2BBe1 cells (VC). Theseresults indicate that all five shRNA/BCRP clones inhibit production ofBCRP mRNA.

For Western blotting, whole cell lysates were collected, the proteincontent quantified, and then 40 μg of protein was loaded and separatedon an 8% Precise® SDS-PAGE protein gel. After separation, proteins weretransferred to a PVDF membrane and immunoblotted. The primary BCRPantibody was a 1:200 dilution of mouse anti-BCRP (Sigma Cat #B7059, Lot#086K177). PVDF membranes were incubated with the primary antibodyovernight at 4° C. After immunoblotting, membranes were exposed to a1:15,000 dilution of goat anti-mouse HRP-conjugated antibody (Zymax Cat#81-6520, Lot #50899722) for 1 hour at room temperature. Staining wasvisualized by the addition of a chemiluminescent horseradish peroxidasesubstrate, using a luminometer. Western blot analysis indicated adecrease in the amount of BCRP present in the shRNA/BCRP clone cells ascompared to C2BBe1 vector control (VC) cells (FIGS. 10 and 11).

RNA production in shRNA/BCRP clone #801 cells was also examined byRT-PCR to determine whether inhibition of BCRP mRNA was stable (FIG.12). The results indicate that mRNA knockdown steadily increased betweenpassages 5 and 20.

2. Functional Characterization of BCRP in Knockdown Clone Cells.

Each of the five shRNA/BCRP knockdown clone cells were seeded ontranswells for three weeks for the transport assay. E3S(Estrone-3-sulfate) a BCRP substrate, was used for the permeabilityassay assessing BCRP function in shRNA/BCRP clone cells. Clones 798,799, 800, and 802 were assayed after 9 passages. Clone 801 was found togrow somewhat faster, so was passaged 13 times prior to the transportassay while waiting for the other clones to grow. Bidirectionaltransport experiments were the same as described in Example 1 and wereconducted using a the QC solution of Example 1 containing 10 μM E3S inaddition to 10 μM digoxin, 10 μM propranolol and 10 μM atenolol, for 2hours. Result indicated that the efflux ratio of Papp for E3S wassignificantly reduced (from 0.91 to 14.42) compared to control cells,which suggests that BCRP activity was inhibited in shRNA/BCRP clonecells (Table 7). Clone #801 showed the highest inhibition for BCRPfunction in E3S transport (96.43%).

TABLE 7 Transport of E3S (Estrone-3-sulfate) in shRNA/BCRP clone cells.Papp values are in units of 10⁻⁶ cm/sec calculated as described inExample 1. Efflux shRNA/BCRP of E3S clone cells efflux number A-B B-Aratio #798 0.86 12.46 14.42 #799 0.87 8.88 10.23 #800 0.82 2.67 3.28#801 0.34 0.31 0.91 #802 0.77 4.38 5.70 Control cell 0.39 10.09 25.87

Atenolol and propranolol (10 μM) were used as passively permeablereference standards for the permeability assay. The permeability assaywas conducted on the same cell monolayers used for the assay of E3Sefflux. Atenolol and propranolol Papp values were not obviouslydifferent from that observed for vector control cells (described inExample 7), implying that BCRP knockdown did not affect thetranscellular passive diffusion pathway (Table 8). Atenolol Papp inclone #801 cells was much lower than vector control cells. TEER values,determined before the permeability assay, were also similar to controlcells except clone #801 cells, which, unexpectedly, had a TEER value 5to 10-fold higher.

TABLE 8 Efflux of reference compounds in cell monolayer of shRNA/BCRPclone cells. Papp values are in units of 10⁻⁶ cm/sec calculated asdescribed in Example 1. shRNA/BCRP clone cells Atenolol Propranolol TEERnumber (A-B) (A-B) (ohm/cm²) #798 0.22 22.68 189 #799 0.21 19.76 173#800 0.18 24.27 172 #801 0.08 25.26 3162 #802 0.24 24.21 317 Vectorcontrol 0.20 19.50 4773. Effect of BCRP Knockdown on Expression and Function of OtherTransporters (MRP2 and P-gp)).

MRP2, P-gp and BCRP are very important transporters located on the cellsurface of C2BBe1 cells. Expression and function of these transporterswere studied in shRNA/BCRP clone cells.

3.1 Expression of mRNA of MRP2 and P-gp in shRNA/BCRP Clone Cells

To determine the effect of BCRP knockdown on expression of P-gp andMRP2, mRNA and protein levels were further studied in clone #801 cellsand compared to clone #799 cells (see FIGS. 13 and 14). Results showedthat P-gp mRNA levels appeared to be higher in clone #801 cells than in#799 and control cells. In contrast, MRP2 mRNA levels were reduced inclone #801 cells passaged 20 times (FIG. 13).

3.2 Functional Characterization of P-gp in shRNA/BCRP Clone Cells

Based on the P-gp mRNA expression profile results, P-gp function wasexamined in shRNA/BCRP clone cells by determining the digoxin effluxratio across 21-day old monolayers plated into 12-well Transwell®transport plates. Experimental conditions were the same as thosedescribed in Example 1. The assay was carried out after each clone hadbeen passaged 9 times. The digoxin efflux ratios were 72.67, 64.47 and54.26 for clones #799, 800, 801 and parental cell monolayers,respectively, indicating higher efflux of this P-gp-specific substrateacross monolayers of BCRP-knockdown cells. The results are presented inTable 9. The results suggest that P-gp function may be enhanced inshRNA/BCRP-clones #799, 800 and 801.

TABLE 9 Transport of digoxin by shRNA/BCRP clone cells shRNA/BCRP Effluxof digoxin clone cells number A-B B-A efflux ratio #798 0.33 7.12 21.43#799 0.11 8.11 72.67 #800 0.06 3.76 64.47 #801 0.05 2.69 54.26 #802 0.223.44 15.77 Control cell 0.20 3.69 18.65

To confirm that P-gp activity was increased in shRNA/BCRP cells, assuggested by the results of the digoxin transport assay, the calcein-AMuptake assay described in Example 1 was repeated with shRNA/BCRP clonecells. Calcein-AM efflux is not mediated by BCRP. Therefore, anyincrease in calcein-AM uptake by the cells can be attributed to P-gpinhibition. The cells were plated in 96-well tissue culture plates andcultured for 48 hours. After 48 hours, cell culture media was removedand the cells were washed with phosphate buffered saline solution (PBS).After washing, cells were incubated with 1 μM calcein-AM for 30 minutes.Parental cells treated with Cyclosporin A, an established P-gpinhibitor, were used as a positive control to verify the P-gp inhibitionincreased the intracellular fluorescence in parental cell lines. Afterincubation with calcein-AM, the cells were rinsed with fresh controlbuffer and fluorescence was measured using a Fluo-star fluorescenceplate reader (BMG Lab Technologies, NC) with excitation and emissionfilters set at wavelengths of 485 nm and 538 nm, respectively. The assaywas carried out after each clone had been passaged 9 times. As shown inTable 10, clones #799, 800, 801 and 802 showed significant functionalinduction of P-gp activity that was comparable to vector control cells.

TABLE 10 Transport of calcein-AM by shRNA/BCRP cells shRNA/BCRP RFU/μgof % decrease of Calcein-AM clone cell number protein compared to vectorcontrol #798 10.13 0.33 #799 6.99 31.18 #800 4.26 58.07 #801 1.08 89.37#802 6.76 33.42 vector control 10.16

Clone #801 showed the highest P-gp functional activity in the calcein-AMassay. A calcein-AM assay was conducted using shRNA/BCRP clone #801cells from a higher cell passage, passage 20, to determine whether theP-gp inductive effect, suggested by the results in Table 10, persistedover numerous cell passages. Results showed that after cell passage 20the activity of P-gp in #801 cells was increased up to 73% compared tovector control cells, results are normalized for total protein. (Table11).

TABLE 11 Transport of calcein-AM by shRNA/BCRP clone #801 cells atpassage 20 Normalized Normalized Mean % Increase over Mean Fluorescencein the Vector Control Cell line/Passage # Fluorescence SD Presence ofCSA SD cells 801/p20 5.08 0.50 72.31 1.51 72.65 Vector Control 23.924.38 62.86 4.34 — cells4. Further Characterization of shRNA/BCRP Clone #801 Cells

While monitoring the growth rates of the various BCRP knockdown clonesproduced by lentiviral transduction, we noted that one clone, #801,started growing at an accelerated rate after being passaged about 15times in vitro. Because the parental C2BBe1 cells take approximately 3weeks to form monolayers suitable for drug transport assays, weinvestigated whether or not clone #801 would mature and form barrierproperties suitable for drug transport assays sooner than the parentalcell line and/or the vector control line.

4.1 Barrier Property of shRNA/BCRP Clone #801 Cells

First, TEER was determined for this cell line (Table 12) at varioustimes after plating into Transwells®. Results showed TEER values wereover 900 Ohm/cm² after 6 days, indicating that tight cell monolayersdevelop rapidly on Transwell® devices.

TABLE 12 TEER for shRNA/BCRP clone #801 cells as determined after 3, 6,10, 15, 20 and 25 days of seeded on transwell. Day after seeded ontrasnwell 3 6 10 15 20 25 TEER (ohm/cm²) 273 979 1383 1464 1934 2736

4.2 Transport of E3S, Digoxin and Permeability Reference Compounds inshRNA/BCRP Clone #801 Cells

As noted, after cell passage 20 shRNA/BCRP clone #801 was found to growfaster and present TEER values much higher than the other four clones.In addition, RT-PCR results suggested that the expression of P-gp mRNAand protein was higher than in control cells (Table 9), Therefore, thetransport of digoxin, which is a substrate of P-gp was conducted in theshRNA/BCRP clone #801 cell monolayers (see Table 13) to determinewhether the apparent increase in P-gp mRNA levels was reflected inincreased efflux transporter function. Transport of E3 S, passivelyabsorbed reference compounds and TEER were also determined at same time.

As shown in Table 13, the digoxin efflux ratio for shRNA/BCRP clone #801cell monolayers was higher than for the wild-type C2BBe1 control cells.Similar results were obtained using the calcein-AM assay (Table 12). TheTEER values of clone #801 monolayers were about 5 times higher thanthose of the wild-type C2BBe1 control cells. The E3S efflux ratio incontrast was still suppressed relative to the parental cell linesindicating that BCRP functional knockdown persisted. The Papp values ofthe reference compound atenolol and propranolol were low and high,respectively, preserving the same rank order as in the wild-type C2BBe1control cells.

TABLE 13 Transport of digoxin, E3S and reference compounds by shRNA/BCRP#801 clone cells 22 and 25 days after seeding on transwells 22 day 25day Control wt KD Control wt KD C2BBe1 #801 C2BBe1 #801 ER of Digoxin18.65 54.26 15.38 42.46 ER of E3S 25.58 0.91 23.06 1.45 TEER 477 3162490 2225 Papp Atenolol 0.13 0.05 0.12 0.08 A-B/×10⁶ cm/sec PappPropranolol 17.2 15.5 18.2 13.6 A-B/×10⁶ cm/sec

4.3 Stability of Expression and Function of P-gp and MRP2 in shRNA/BCRPClone #801 Cells.

To determine the stability of the effect of BCRP knockdown on theexpression of other transporter genes, mRNA levels of P-gp and MRP2 aswell as protein levels were determined in shRNA/BCRP clone #801 cellsfrom cell passage 10 to 20. The results indicated that expression ofP-gp mRNA was increased and MRP2 was reduced at cell passage 20 (seeFIGS. 13 and 14).

Bidirectional transport measurements of E3S, a BCRP substrate, anddigoxin, a P-gp substrate as well as TEER measurements were conducted atsame time in the same cell cultures. The digoxin efflux (see Table 14)was higher than vector control cell (25.13) in clone #801 cells frompassage 18 to 25, and the highest efflux ratio was observed for cellpassage 25 (114.98).

TABLE 14 Effect of BCRP knockdown on digoxin transport by shRNA/BCRPclone #801 cells at different cell passages. Vector Cell passage 9 18 25control Efflux ratio (Papp (B-A/A-B)) 20.43 14.57 114.98 25.13 TEER(ohm/cm²) 347 439 1589 296

The E3S efflux ratio (see Table 15) was kept below 3.0 from cell passage9 to 25 in clone #801 cells compared to vector control cells (13.96).The TEER values of the clone #801 cell monolayers increased from 347 to1589 ohms/cm² from cell passage 9 to 25, and was higher than vectorcontrol cells.

TABLE 15 Effect of BCRP knockdown on E3S transport by shRNA/BCRP clone#801 cells at different cell passages. Vector Cell passage 9 18 25control Efflux ratio (Papp (B-A/A-B)) 0.59 1.54 0.52 13.96 TEER(ohm/cm²) 347 565 1589 296

EXAMPLE 6 Evaluation of the Role of P-up, MRP2, and BCRP in DrugAbsorption

The following prophetic example provides an experimental approach todetermine the identity of a transporter(s) responsible for handling adrug of interest within the gastrointestinal tract of animals. Thisexperimental approach comprises a combination of (a) cell lines (e.g.,MDCK) expressing single (transfected) transporters, (b) C2BBe1 cellswith selectively knocked-down transporters and (c) chemical inhibitorsof transporters. Together, these tools will facilitate the determinationof the identity of the transporters involved in a drug transport processwith a high degree of certainty.

Experiment 1. Efflux transporter(s) involvement. In this experiment,bidirectional transport of a test compound can be screened in C2BBe1WTmonolayers, and the PappB-A/PappA-B ratio (i.e., efflux ratio, ER) canbe calculated. Based on the results of these experiments, subsequenttests, as described below, can be carried out.

There are at least two possible outcomes for this experimental approach.In the first possible outcome, an ER<3 indicates that permeation doesnot involve membrane efflux transport proteins. Under this scenario, noadditional testing for transporters would be required, except forpossible confirmatory experiments. In the second possible outcome, anER≧3 indicates that permeation involves at least one membrane effluxtransport protein present in C2BBe1 cells (e.g., P-gp, MRP2 or BCRP). Toidentify which of these transporter(s) is involved in the efflux, thefollowing experiments will be performed.

Experiment 2. Confirmation of P-gp involvement. To determine if P-pgplays a role in handling the drug of interest, bidirectional permeationexperiments, as described and exemplified herein, can be carried out.Such bidirectional permeation experiments will be used to screen thefollowing permutations: (a) test compound alone in C2BBe1 WT monolayers;(b) test compound alone in C2BBe1 P-gp-knockdown monolayers; (c) testcompound with MRP inhibitor (e.g., MK571) in C2BBe1 P-gp-knockdownmonolayers; (d) test compound with BCRP inhibitor (e.g., FTC) in C2BBe1P-gp-knockdown monolayers; (e) test compound alone in MDR1-MDCKmonolayers; and, (f) test compound with a P-gp inhibitor (e.g., CsA) inMDR1-MDCK monolayers

Experiment 3. Confirmation of BCRP involvement. To determine if BCRPplays a role in handling the drug of interest, bidirectional permeationexperiments, as described and exemplified herein, can be carried out.Such bidirectional permeation experiments will be used to screen thefollowing permutations: (a) test compound alone in C2BBe1 WT monolayers;(b) test compound alone in C2BBe1 BCRP-knockdown monolayers; (c) testcompound alone in BCRP-MDCK monolayers; and, (d) test compound with BCRPinhibitor (e.g., FTC) in BCRP-MDCK monolayers.

Experiment 4. Confirmation of MRP2 involvement. To determine if MRP2plays a role in handling the drug of interest, bidirectional permeationexperiments, as described and exemplified herein, can be carried out.Such bidirectional permeation experiments will be used to screen thefollowing permutations: (a) test compound alone in C2BBe1 WT monolayers;(b) test compound alone in C2BBe1 MRP2-knockdown monolayers; (c) testcompound alone in MRP2-MDCK monolayers; and, (d) test compound with MRP2inhibitor (e.g., MK571) in MRP2-MDCK monolayers

The following is an example of the approaches described above using twocell lines to identify whether or not compounds are substrates for P-gpor BCRP or both. Each compound was assayed in a bidirectional cellmonolayer assay as described in Example 1. Concentrations of theselected compounds were determined by LC/MS/MS, as described in Example1, by monitoring the mass transitions recorded in column 2 of Table 16.The cell monolayers consisted either of the parental C2BBe1 cells orshRNA/BCRP clone #801 cells that express only P-gp to a significantextent.

TABLE 16 Comparison of the bidirectional efflux ratios of selected P-gpsubstrates and non-substrates across monolayers of shRNA/BCRP clone #801cells or parental C2BBe1 cells. Efflux Ratio in Efflux Ratio Mass KDClone in C2BBe1 P-gp Compound Transition 801 cells cells Substrate?Antipyrine 189.20/56.10  0.92 0.84 No Etoposide 589.30/229.00 11.5313.67 Yes Sulfasalazine 329.00/284.90 0.22 15.45 No Furosemide329.00/284.90 1.38 21.76 No Diphenhydramine 256.30/167.10 1.64 2.41 NoDesipramine 267.20/72.00  6.84 5.59 Yes

Antipyrine is not a substrate for P-gp because it does not show effluxin either cell line. Etoposide is a substrate because it shows efflux inboth cell lines to a similar extent. Sulfasalazine is not a P-gpsubstrate because it is not effluxed by the shRNA/BCRP clone#801 cellseven though it is effluxed by the C2BBe1 cells. This result suggeststhat sulfasalazine is a substrate for either BCRP and/or MRP2 which areexpressed at high levels in the C2BBe1 cells, but not in shRNA/BCRPclone #801 cells. In fact, sulfasalazine is known to be a BCRP substrate(Table 1). In fact, the efflux ratio of sulfasalazine in the shRNA/BCRPclone #801 cells is 0.22, suggesting that these cells possess an uptaketransporter for this compound whose activity is revealed by BCRPknockdown. Furosemide shows a similar pattern to sulfasalazine, butwithout evidence of the presence of an uptake transporter.Diphenhydramine is not highly effluxed by either cell line, whiledesipramine is, indicating that desipramine is a P-gp substrate. Theseresults could be extended and confirmed by repeating the above studyusing shRNA/P-gp clones #83 or #85 and demonstrating that the efflux ofthe putative P-gp substrate is much lower across monolayers of the P-gpknockdown cells, while sulasalazine and furosemide still show highefflux.

EXAMPLE 7 Transduction of shRNA of Non-Target shRNA Control TransductionParticles in C2BBe1 Cells

In some cases, parental C2BBe1 cells may not be appropriate positivecontrols for studying transport function of the knockdown cell lines,because of differences in media composition, growth rates and otherpossible effects produced by the presence of lentiviral gene products inthe cells. When conducting experiments using shRNA clone cells, propercontrols are a key element of experimental design to permit accurateinterpretation of knockdown results and provide assurance of thespecificity of responses observed. MISSION® non-target shRNA controlvector containing SEQ ID NO: 27 available from Sigma-Aldrich, St. Louis,Mo., is a useful negative control that will activate the RISC and theRNAi pathway, but does not target any human genes.

Non-target shRNA MISSION® shRNA Lentiviral Transduction Particles (cat.no. SHC002V) were obtained from Sigma-Aldrich, St. Louis, Mo. and usedto generate C2BBe1 cell lines transduced with non-target shRNA.Lentiviruses containing the non-target shRNA were used to transformC2BBe1 cells according to the manufacturer's protocols. In brief, C2BBe1cells were seeded into a 96 well plate at 1.6×10⁴ cells per well in cellculture media (90% DMEM+10% FBS), and incubated at 37° C. for 24 hoursprior to transduction. On the day of transduction, media was removedfrom the wells and lentiviral particles were added to the wells at 0.5,1.0, or 5.0 MOI (multiplicity of infection) and incubated with the cellsfor 24 hours at 37° C. Media containing unbound lentiviral particles wasremoved after this incubation, and fresh media was supplied. At 48 hourspost-transduction, selective medium containing 10 Mg/mL puromycin wasadded and changed every 3-4 days thereafter to select for transducedcells. Positive cells were prepared as individual cell clones, thengrown and evaluated in the various functional assays described herein.Clones shown to express P-gp, BCRP and MRP2 at levels similar oridentical to that in the parental cell lines as well as possessingsimilar passive permeability properties were used as positive controlsin some of the experiments described in Examples 4 and 5.

Experimental Results of Testing Control Cells:

C2BBe1 cells were transformed with a shRNA lentiviral vector containingSEQ ID NO. 27 as described in Example 2. To determine the optimal degreeof multiplicity of infection (MOI), three MOIs were used to transducethe cells (0.5, 1.0 and 5.0). The molecular and functional assays weredone as describe in Examples 3 to 5 for the same assays in geneknockdown cells. Expression of BCRP, MRP2 and P-gp in shRNA vectorcontrol cells was examined by RT-PCR and Western blot, which wereconducted by the same methods used to examine knockdown cells. Theresults indicate that the vector control cells still express thetransporter mRNAs (Table 17).

TABLE 17 Expression of BCRP, MRP2 and P-gp mRNA in shRNA vector control(VC) cells. Ratio of trasnporter/B-actin Cell Group MOI P-gp BCRP MRP-2C2B Bel — 0.62 0.70 1.54 Vector control 0.5 0.66 0.73 1.53 1.0 0.97 0.782.04 5.0 0.85 0.66 1.74

Comparison of Typical Transport Properties Between shRNA Vector Controland Parental C2BBe1 Cells.

Vector control and C2BBe1 cell lines were cultured on 12-well Transwell®plates for 21 days prior to conducting the transport assays indicated inTable 18. The assays were performed under conditions described inExample 1. The results indicate that the vector control cells formmonolayers on Transwell® devices with barrier properties suitable for invitro drug transport assays and that they express efflux activityagainst digoxin, a P-gp substrate, and estrone-3-sulfate, a BCRPsubstrate (Table 18).

TABLE 18 Comparison of typical transport properties between shRNA vectorcontrol (MOI = 1) and parental C2BBe1 cell. shRNA vector Parental Papp &Substrate control C2BBe1 Papp A-B (E3S) (×10⁶ cm/sec) 0.74 0.63 Papp B-A(×10⁶ cm/sec) 10.36 10.69 Ratio of Papp(B-A/A-B) 14 17 Papp A-B(Digoxin) (×10⁶ cm/sec) 0.33 0.2 Papp B-A (×10⁶ cm/sec) 8.24 3.69 Ratioof Papp(B-A/A-B) 25 19 Papp A-B (Atenolol) (×10⁶ cm/sec) 0.39 0.35 PappA-B (Propranolol) (×10⁶ cm/sec) 13.88 22.4 TEER (ohms · cm²) 446 477

The present invention is not limited to the embodiments described andexemplified above, but is capable of variation and modification withinthe scope of the appended claims.

1. A host cell transformed with a vector comprising a nucleic acidsequence encoding a nucleic acid molecule for inhibiting expression ofp-glycoprotein, wherein the nucleic acid sequence comprises SEQ ID NO:1, 2, 3, 4, or
 5. 2. The host cell of claim 1, wherein the cellexpresses p-glycoprotein.
 3. The host cell of claim 1, wherein the cellis an intestinal epithelial cell.
 4. A cell culture comprising the hostcell of claim
 3. 5. The host cell of claim 1, wherein said host cell hasbeen deposited with the American Type Culture Collection and assignedaccession number PTA-8752 or PTA-8753.
 6. The host cell of claim 1,wherein the cell is a Caco-2 cell.
 7. The host cell of claim 1, whereinthe cell is derived from a Caco-2 cell.
 8. The host cell of claim 1,wherein the cell is a C2BBe1 cell.
 9. A kit for determining theabsorption characteristics of a chemical compound, comprising the cellof claim
 1. 10. A nucleic acid sequence comprising SEQ ID NO: 1, 2, 3,4, or
 5. 11. A nucleic acid encoded by the nucleic acid sequence ofclaim
 10. 12. A vector comprising a nucleic acid sequence encoding anucleic acid molecule for inhibiting expression of p-glycoprotein,wherein the nucleic acid sequence comprises SEQ ID NO: 1, 2, 3, 4, or 5.